Aptamers to PDGF and VEGF and Their Use in Treating PDGF and VEGF Mediated Conditions

ABSTRACT

Aptamers that bind PDGF and aptamers that bind VEGF are provided. In addition, aptamer constructs comprising a PDGF aptamer and a VEGF aptamer are provided. Pharmaceutical compositions comprising the aptamers and aptamer constructs are provided, as well as methods of treating conditions using the aptamers and aptamer constructs.

RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 15/602,377, filed May 23, 2017. U.S. application Ser. No. 15/602,377is a continuation application of U.S. application Ser. No. 15/189,159,filed Jun. 22, 2016, now U.S. Pat. No. 9,701,967. U.S. application Ser.No. 15/189,159 is a divisional application of U.S. application Ser. No.14/381,679, filed Aug. 28, 2014, now U.S. Pat. No. 9,410,156. U.S.application Ser. No. 14/381,679 is a national stage entry ofinternational application no. PCT/US2013/034493, filed on Mar. 28, 2013(WO 2013/149086). International application no. PCT/US2013/034493 claimsthe benefit of U.S. provisional application Ser. No. 61/616,881, filedMar. 28, 2012; U.S. provisional application Ser. No. 61/648,394, filedMay 17, 2012; U.S. provisional application Ser. No. 61/719,354, filedOct. 26, 2012; and U.S. provisional application Ser. No. 61/722,099,filed Nov. 2, 2012. Each of the applications referenced above is herebyincorporated by reference in their entirety for all purposes as if fullyset forth herein.

FIELD OF THE INVENTION

The present disclosure relates generally to the field of nucleic acidsand more particularly to aptamers capable of binding to platelet-derivedgrowth factor (PDGF) and aptamers capable of binding to vascularendothelial growth factor (VEGF). In some embodiments, such aptamers areuseful as therapeutics for preventing, treating, and/or amelioratingproliferative disorders, including, but not limited to, atherosclerosis,macular degeneration, fibrosis, cancer, and other disorders in whichPDGF and/or VEGF has been implicated. In some embodiments, the presentdisclosure relates to aptamer constructs that are capable of binding toVEGF and PDGF, either simultaneously or in a mutually exclusive manner,and are useful as therapeutics.

Incorporated by reference herein in its entirety is the Sequence Listingentitled “Sequence Listing_0057_57PCT_ST25”, created Jan. 12, 2016, sizeof 883 kilobytes.

BACKGROUND

The following description provides a summary, of information, and is notan admission that any of the information provided or publicationsreferenced herein is prior art to the present disclosure.

Platelet-derived growth factors (PDGF-A, -B, -C and -D) are ubiquitousmitogens and chemotactic factors for many connective tissue cells(Fredriksson, L., et al. (2004) Cytokine Growth Factor Rev. 15(4): 197).PDGFs occur as disulfide-linked dimers and contain a cysteine-knot-foldgrowth factor domain that functions through binding to PDGF receptors αand β on the surface of a cell (Claesson-Welsh, L., et al. (1989) Proc.Natl. Acad. Sci. USA 86:4917). PDGF binding induces receptordimerization, which leads to autophosphorylation at intracellulartyrosine residues (Claesson-Welsh, J. (1994) Biol. Chem. 269:32023).PDGF-BB is involved in several proliferative disorders, includingatherosclerosis, fibrosis, macular degeneration, and cancer (Ostman, A.,et al. (2001) Adv. Cancer Res. 80:1; Appelmann, I., et al. (2010) RecentResults Cancer Res. 180:51; Trojanowska, M., et al. (2008) Rheumatology(Oxford) 47(Suppl 5):2; Rutherford et al. (1997) Atherosclerosis 130:45;Smits et al. (1992) Am. J. Pathol. 140:639; Heldin et al. (1991)Endocrinology 129:2187; Floege and Johnson (1995) Miner. ElectrolyteMetab. 21:271; Raines et al. (1990) Experimental Pharmacology, PeptideGrowth Factors and Their Receptors, Sporn & Roberts, pp. 173-262,Springer, Heidelberg.).

VEGF is a secreted disulfide-linked homodimer that selectivelystimulates endothelial cells to proliferate, migrate and producematrix-degrading enzymes, all of which are processes required for theformation of new blood vessels (Conn, G., et al. (1990) Proc. Natl.Acad. Sci. USA 87:1323; Ferrara, N. et al. (1989) Biochem. Biophys. Res.Commun. 161:851; Gospodarowicz, D., et al. (1989) Proc. Natl. Acad. Sci.USA 86:7311; Pepper, M. S., et al. (1991) Biochem. Biophys. Res. Commun.181:902; Unemori, E. N., et al. (1992) J. Cell. Physiol. 153:557). Inaddition to being the only known endothelial cell-specific mitogen, VEGFis unique among angiogenic growth factors in its ability to induce atransient increase in blood vessel permeability to macromolecules(Dvorak, H. F., et al. (1979) J. Immunol. 122:166; Senger, D. R., et al.(1983) Science 219:983; Senger, D. R., et al. (1986) Cancer Res.46:5629). Increased vascular permeability and the resulting depositionof plasma proteins in the extravascular space facilitate new vesselformation by providing a provisional matrix for the migration ofendothelial cells. Hyperpermeability is indeed a characteristic featureof new vessels (Dvorak, H. F., et al. (1995) Am. J. Pathol. 146:1029).Furthermore, compensatory angiogenesis induced by tissue hypoxia is alsomediated by VEGF (Levy, A. P., et al. (1996) J. Biol. Chem. 271:2746;Shweiki, D., et al. (1991) Nature 359:843). The identification of VEGFas a hypoxia-inducible protein, along with the complementary observationthat hyperoxia causes suppression of VEGF expression, provides anappealing mechanism for matching oxygen demand with vascular supply(Benjamin, L. E., et al. (1999) J. Clin. Invest. 103:159; Alon, T., etal. (1995) Nat. Med. 1:1024).

Several isoforms of VEGF protein occur as a result of alternativesplicing of the eight exons of the gene that encodes VEGF (Eming, S. A.,et al. (2006) J. Invest. Dermatol. Symp. Proc. 1:79). The most prevalentisoforms are VEGF-121, VEGF-165 and VEGF-189. Proteolytic processing ofVEGF can generate additional isoforms. VEGF-165 can be cleaved byplasmin between Arg-110 and Ala-111 to generate VEGF-110, which isfunctionally equivalent to VEGF-121 (Keyt, B. A., et al. (1996) J. Biol.Chem. 271:7788). VEGF-189 can be cleaved by urokinase within the exon 6domain and then can be cleaved further by plasmin to generate VEGF-110(Plouet, J., et al., (1997) J. Biol. Chem. 272:13390). In addition, asubset of matrix metalloproteases (MMPs), including MMP-3, -7, -9 and-19, are capable of cleaving VEGF-165 and VEGF-189 in sequential stepsto generate VEGF-113, which is functionally equivalent to VEGF-110.Therefore, the relative abundance of matrix-bound and diffusible formsof VEGF in a given tissue is determined by the combination ofalternative splicing and proteolytic processing that occurs in the cellsof the tissue (Ferrara, N., et al. (2006) Retina 26:859).

Age-related macular degeneration (AMD) remains the leading cause ofblindness in people over 55 years of age. The disease is characterizedby the formation of insoluble deposits called drusen within the macula,the part of the retina that has the highest density of photoreceptorsand is involved in central vision. In the initial stages of AMD, thedeposits are avascular and the disease generally progresses slowly.However, in about 10% of the patients, this so-called “dry” form of AMDbecomes vascularized and turns into the “wet” form of AMD, during whichthe disease becomes more progressive and vision deteriorates at a fasterrate. In many cases, the progression from blurriness of central visionto virtual blindness occurs in less than two years. In the advancedstage of the disease, the exudative or wet form of AMD, new bloodvessels penetrate from the choriocapillaris into the central part of theretina (macula), occluding central vision. In the United States, theprevalence of wet AMD is about 1.8 million and is expected to increaseto close to 3 million by 2020. The incidence of wet AMD in the UnitedStates is about 210,000 people each year.

Recently, AMD has been treated by blocking VEGF-mediated induction ofangiogenesis and blood vessels leakiness by direct injection into theeye of high-affinity antagonists that bind to VEGF, preventinginteraction of VEGF with its cell-surface receptors on endothelialcells.

There is considerable evidence that dual inhibition of VEGF and PDGF-Bsignaling leads to more efficient blocking of angiogenesis coupled withregression of new blood vessels. For example, clinical evidence suggeststhat dual inhibition of VEGF and PDGF-B can achieve a more completeinhibition of ocular angiogenesis in AMD patients. An aptamer inhibitorof PDGF-B (E10030), originally discovered at NeXstar Pharmaceuticals(Green, L. S., et al. (1996) Biochemistry 35:14413; U.S. Pat. Nos.6,207,816; 5,731,144; 5,731,424; and 6,124,449), is being developed byOphthotech Corporation as a treatment for AMD. E10030 (Fovista®) is aDNA-based modified aptamer that binds to PDGF-AB or PDGF-BB with a K_(d)of approximately 100 pM and inhibits the functions of PDGF-B both invitro and in vivo.

In a Phase 1 study, anti-PDGF therapy with E10030 tested in combinationwith Lucentis® anti-VEGF therapy resulted in vision gain of three linesin 59% of treated patients after 12 weeks of therapy. This is aconsiderably higher percentage of patients with improved visual acuitycompared to the 34-40% observed historically with Lucentis alone. Inaddition, the combination treatment was accompanied with markedneovascular regression in all study participants. Enhanced efficacy withcombination treatment was recently corroborated in a phase 2 study of449 patients with wet AMD. Patients receiving the combination of Fovista(1.5 mg) and Lucentis gained a mean of 10.6 letters of vision at 24weeks, compared to 6.5 letters for patients receiving Lucentismonotherapy (p=0.019), representing a 62% additional visual acuitybenefit.

SUMMARY

The present disclosure provides aptamers that bind to platelet-derivedgrowth factor B (PDGF-B, including PDGF-BB and PDGF-AB), aptamers thatbind vascular endothelial growth factor (VEGF, including VEGF-121 andVEGF-165), and aptamer constructs comprising an aptamer that bindsPDGF-B and an aptamer that binds VEGF. The disclosed aptamers andaptamer constructs are useful as therapeutics for preventing, treating,and/or ameliorating proliferating diseases or conditions, including butnot limited to, atherosclerosis, macular degeneration, fibrosis,diabetic retinopathy, and cancer, and/or other diseases or conditions inwhich PDGF and/or VEGF is implicated. In various embodiments, theaptamer constructs are capable of binding to each of VEGF and PDGF-Bindependently and/or VEGF and PDGF-B simultaneously. Included arepharmaceutical compositions or formulations comprising a PDGF aptamer, aVEGF aptamer, or a VEGF/PDGF-B aptamer construct, or a pharmaceuticallyacceptable salt of any of the foregoing, and at least onepharmaceutically acceptable carrier. Such compositions can be preparedin any suitable pharmaceutically acceptable dosage form.

In another aspect, the present disclosure provides methods forpreventing, treating, and/or ameliorating a disease or conditionmediated by PDGF and/or VEGF. In some embodiments, a method comprisesadministering a PDGF aptamer, a VEGF aptamer, and/or a VEGF/PDGF-Baptamer construct, or pharmaceutical compositions comprising any ofthese, to a subject, such as a mammal. In some embodiments, the subjectis a human. Specifically, methods for treating, preventing, and/orameliorating fibrosis, atherosclerosis, macular degeneration, diabeticretinopathy, and/or cancer are provided. In some embodiments, a diseaseor condition mediated by PDGF and/or VEGF is one in which PDGF and/orVEGF activity may directly or indirectly contribute to the disease orcondition. Such diseases or conditions include, but are not limited to,fibrosis, atherosclerosis, macular degeneration, diabetic retinopathy,and cancer. In some embodiments the disease or condition to be treated,prevented, and/or ameliorated is age-related macular degeneration (AMD),diabetic retinopathy, or other ocular diseases, such as glaucoma,chronic dry eye, AIDS-related vision loss, amblyopia, hemianopia,retinal vein occlusions, trachoma, keratoconus, chorioretinalinflammation, central serous retinopathy, uveitis, retinitis,hypertensive retinopathy, retinal dystrophy, etc. In some embodiments,the disease or condition to be treated, prevented, and/or ameliorated isrenal fibrosis or renal cancer.

In some embodiments, aptamers and aptamer constructs disclosed hereinhave potential applications ranging from biomarker discovery anddiagnostics (Ostroff, R. M., et al. (2010) PLoS One 5:e15003; Mehan, M.,et al. (2012) PLoS One 7:e35157) to histochemistry and imaging (Gupta,S., et al. (2011) Appl. Immunohistochem. Mol. Morphol. 19:273).

In some embodiments, a therapeutic effect (e.g., treating, preventing,and/or ameliorating fibrosis, atherosclerosis, macular degeneration, orcancer, etc.) may be achieved by administering a PDGF aptamer, a VEGFaptamer, and/or a PDGF/VEGF aptamer construct such that the aptamer oraptamer construct is exposed to, and can bind to, PDGF and/or VEGF. Insome embodiments, such binding occurs regardless of the method ofdelivery of the aptamer to the subject being treated. In someembodiments, the therapeutic effect may be achieved by administering thePDGF aptamer, VEGF aptamer, or PDGF/VEGF aptamer construct such that itis exposed to, and binds to, PDGF and/or VEGF and prevents or reducesthe binding of PDGF and/or VEGF to one or more cell receptors.

In some embodiments, the binding of a PDGF aptamer to PDGF-BB or PDGF-ABinterferes with the binding of PDGF-BB or PDGF-AB to the PDGF-αreceptor. In some embodiments, the binding of a PDGF aptamer to PDGF-BBor PDGF-AB interferes with the binding of PDGF-BB or PDGF-AB to thePDGF-β receptor. In some embodiments, a PDGF aptamer to PDGF-BB orPDGF-AB reduces phosphorylation of a PDGF receptor (such as PDGF-αreceptor and/or PDGF-β receptor).

In some embodiments, the binding of a VEGF aptamer to VEGF-121,VEGF-110, VEGF-165, VEGF-189, or another alternatively spliced orfunctionally-active proteolytic fragment of VEGF interferes with thebinding of the growth factor to VEGFR-1 (Fit-1). In some embodiments,the binding of a VEGF aptamer to VEGF-121, VEGF-110, VEGF-165, VEGF-189,or another alternatively spliced or functionally-active proteolyticfragment of VEGF, interferes with the binding of the growth factor toVEGFR-2 (KDR). In some embodiments, a VEGF aptamer reducesphosphorylation of a VEGF receptor (such as VEGF-1 receptor and/orVEGF-1 receptor).

In some embodiments, a PDGF/VEGF aptamer construct reduces the level ofphosphorylation of a PDGF receptor (such as PDGF-α receptor and/orPDGF-β receptor) and reduces the level of phosphorylation of a VEGFreceptor (such as VEGFR-1 and/or VEGFR-2). In some embodiments, a PDGFaptamer, a VEGF aptamer, or a PDGF/VEGF aptamer construct reducessignaling along the signal transduction pathway of a PDGF receptorand/or a VEGF receptor.

In some embodiments, a PDGF aptamer, a VEGF aptamer, or a PDGF/VEGFaptamer construct is administered with one or more additional activeagents. Such administration may be sequential or in combination.

In some embodiments, an in vitro diagnostic method comprises contactinga PDGF aptamer with a sample suspected of comprising PDGF. In someembodiments, an in vivo diagnostic method comprises administering asuitably labeled PDGF aptamer to an individual suspected of having aPDGF-mediated disease or disorder, wherein the labeled PDGF aptamer isdetected for the purpose of diagnosing or evaluating the health statusof the individual. The label used may be selected in accordance with theimaging modality to be used.

In some embodiments, an in vitro diagnostic method comprises contactinga VEGF aptamer with a sample suspected of comprising VEGF. In someembodiments, an in vivo diagnostic method comprises administering asuitably labeled VEGF aptamer to an individual suspected of havingVEGF-mediated disease or disorder, wherein the labeled VEGF aptamer isdetected for the purpose of diagnosing or evaluating the health statusof the individual. The label used may be selected in accordance with theimaging modality to be used.

In some embodiments, an in vitro diagnostic method comprises contactinga PDGF/VEGF aptamer construct with a sample suspected of comprising PDGFand/or VEGF. In another aspect, the present disclosure provides an invivo diagnostic method comprising obtaining a suitably labeled PDGF/VEGFaptamer construct, injecting the labeled PDGF/VEGF aptamer constructinto an individual suspected of having a PDGF/VEGF-mediated disease ordisorder, and detecting the labeled PDGF/VEGF aptamer construct for thepurpose of diagnosing or evaluating the health status of the individual.The label used may be selected in accordance with the imaging modalityto be used.

In some embodiments, the present invention provides aptamer constructscomprising a PDGF aptamer and a VEGF aptamer.

In some embodiments, the present disclosure provides two discreteco-crystal structures, solved at resolutions of 2.2 Å and 2.3 Å, eachcontaining two copies of an aptamer bound to PDGF-BB.

In some embodiments, the present disclosure provides an aptamer thatefficiently binds to a protein predominantly through hydrophobicinteractions.

In some embodiments, the present disclosure provides an aptamer-proteincomplex, wherein the aptamer binds to the protein substantially throughhydrophobic interactions.

In some embodiments, the present disclosure provides an aptamer capableof forming a co-crystal complex with a protein target, wherein thecomplex comprises fewer than 7 hydrogen bonds.

In some embodiments, the present disclosure provides an aptamer capableof forming a co-crystal complex with a protein target, wherein thecomplex comprises a pseudoknot domain involving 16 nucleotides or less.

In some embodiments, the present disclosure provides an aptamer capableof binding to a protein target, wherein the aptamer binds to the proteintarget with less than or equal to 1 polar contact per 100 Å² ofinterface area, wherein the polar contact comprises one or more hydrogenbonds and one or more charge-charge interactions, and wherein theinterface area is a fraction of the protein surface area occupied by theaptamer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows determination of K_(d) values for three slow off ratemodified aptamers and aptamer E10030. FIG. 1B shows inhibition ofPDGF-BB-stimulated phosphorylation of PDGFRβ by three PDGF aptamersdescribed herein and a scrambled control oligonucleotide. FIGS. 1C and1D show the K_(d) ratios for modified aptamers versus the parentaptamer, 4149-8_130, in which a particular Bn-dU nucleobase has beenreplaced by another modified dU nucleobase, relative to the parentaptamer (a value of 1 indicates the modified aptamer inhibitsphosphorylation of PDGFRβ equally as well as the parent, while avalue >1 indicates the modified aptamer has less potent inhibitoryactivity compared to the parent).

FIG. 2A shows a table of aptamer 4149-8_260 intramolecular contacts andaptamer-PDGF contacts. FIG. 2B shows a representation of nonpolarintramolecular and aptamer-PDGF contacts; and FIG. 2C shows arepresentation of polar intramolecular and aptamer-PDGF contacts; asdescribed in Example 2. Hydrophobic (nonpolar) interactions include π-πinteractions (both face-to-face and edge-to-face aromatic interactions)and van der Waals contact (vW). Polar interactions include hydrogenbonds (dashed lines) and charge-charge interactions (solid lines).Certain aptamer residues (e.g., dC4, dG6, dA9, dC10, dC12, dG13, dC14,dG15, dG22, dC23 and 2′-O-methyl G24) participate in canonical basepairing and base stacking, as shown in B and C, and 2′-O-methyl A11 isextruded.

FIG. 3A shows the consensus sequence for a set of clones from the SELEXpool as determined by 454 pyrosequencing and nucleotide frequency ateach position, and the sequences for six of the clones; and FIG. 3Bshows K_(d) values for modified aptamers based on parent aptamer 4149-8,which were modified as shown, as described in Example 1.

FIG. 4 illustrates certain stereoviews of a PDGF-BB: 4149-8_260 complex,as described in Example 2.

FIG. 5A shows binding affinity of various aptamers for different PDGFdimeric isoforms, in the presence and absence of 200 nM tRNA; and FIG.5B shows an alignment of the amino acid sequences for the mature formsof PDGF-A, -B, -C and -D; as described in Example 3. Amino acid residuesfor PDGF-A that are shown in bold are involved in propeptide binding,and amino acid residues for PDGF-B that are shown in bold are involvedin PDGFRβ binding (Shim et al, (2010) Proc. Natl. Acad. Sci. USA107(25):11307). Box shading indicates residues involved in aptamer4149-8_260 binding to PDGF-B chain 1 (dark shading), and PDGF-B chain 2(light shading).

FIG. 6A shows the K_(d) ratio for modified aptamers made by substitutingeach indicated position in aptamer 4149-8_38 (SED ID NO: 38) with athree-carbon C-3 linker, and K_(d) values for PDGF-BB binding, PDGF-ABbinding, as well as cellular IC₅₀ for five modified aptamers based onparent aptamer 4149-8; and FIG. 6B shows K_(d) ratios for modifiedaptamers based on parent aptamer 4149-8_130 (SEQ ID NO: 130), in which aparticular Bn-dU nucleobase has been replaced by another modified dUnucleobase, relative to the parent aptamer (numbers <1 indicate themodified aptamer has greater affinity than the parent aptamer, andnumbers >1 indicate the modified aptamer has lower affinity than theparent aptamer); as described in Example 1.

FIG. 7A shows a ribbon diagram of the PDGF-BB homodimer bound to aptamer4149-8_260 (SEQ ID NO: 211); and FIG. 7B shows a schematicrepresentation and structural representation of the aptamer conformationwhen bound to a PDGF-B subunit; as described in Example 2. Non-canonicalbase pairs are coded based on the nomenclature of Leontis and Westhof(Leontis N. B. et al. (2003) Curr. Opin. Struct. Biol. 13(3):300). Darkgray=PDGF-B Chain 1; Light gray=PDGF-B Chain 2; Bn=Bn-dU, Pe=Pe-dU,Th=Th-dU.

FIGS. 8A-8N illustrate certain structural features of the PDGF aptamerfrom the crystal structure of the PDGF-BB: 4149-8_260 aptamer complex,as described in Example 2. FIG. 8A illustrates the aptamer structure,showing domains, basepairing and stacking interactions. The stems of theminiknot deviate significantly from B-form DNA due to substantialbuckling and propeller angles as well as helical underwinding. Bn20 isthe hinge that interfaces with the 5′ stem through stacking with U8.FIG. 8B illustrates Stem 1 (S1) end view. FIG. 8C illustrates S1 and L2side views. The modified nucleotides form a hydrophobic cluster withBn2, Bn7 and Bn8 from the 5′ stem interacting with Bn16, Pel7, Th18 andBn20 from the miniknot. Bn8 makes edge-to-face π-π interactions withBn16 and Bn20. The non-canonical dU-dU base pair utilizes H-bonding tothe amide linker of Bn20. FIG. 8D illustrates the details of thenon-canonical base pair between Pe-dU17 and Bn-dU20. FIG. 8E illustratesaromatic interactions stabilizing the base of miniknot S1. FIG. 8Fillustrates a base triple which is formed between the C10-G15Watson-Crick pair in the S1 and L2 nucleotide, A21. The Leontis-Westhofclassification of this base triple is cis Watson-Crick/Watson-Crick,trans Sugar edge/Hoogsteen (Leontis N. B. et al. (2003) Curr. Opin.Struct. Biol. 13:300). The base triple is not planar as there is a 34°propeller twist angle between A21 and G15 as well as considerablebuckling and propeller twisting between the Watson-Crick base pair(Table 5). FIG. 8G illustrates residue mA11, the single extruded base inL1 and the backbone turn. FIG. 8H illustrates an axial view of S2.

FIG. 8I illustrates an axial view of the 5′ stem motif which highlightsthe significant deviation from B-form DNA. The global C1′-C1′ helicalparameters indicate the dU-dU pair is overtwisted (40°) resulting in a˜124° bend in the backbone which flattens to near linear (˜172°) betweenBn-dU1 and Bn-dU2. The significant radial displacement betweenBn-dU7-Bn-dU8 and near zero displacement between Bn-dU2-dA3 results ingreater stacking overlap between bases 2-4 and 6-7. FIG. 8J whichillustrates the non-canonical base pair between Bn-dU2 and Bn-dU8. FIG.8K illustrates the interdomain junction formed by modified nucleotides.FIG. 8L illustrates Bn8 making edge-to-face π-π interactions with Bn16and Bn20 that define the topology of the interdomain junction. Thedeleterious impact of substituting Bn-dU at position 8 of aptamer4149-8_260 (SEQ ID NO: 211) with an iB-dU (SEQ ID NO: 255) is evident inthe space-filling images shown in FIG. 8M and FIG. 8N. Bn8 (FIG. 8M) iscapable of making energetically favorable π-π interactions withneighboring aromatic groups and allows the SOMAmer to pack more tightly.In contrast, iB8 (FIG. 8N) is not aromatic and thus lacks the abilityfor π-stacking interactions with neighboring aromatic groups.Additionally, the iB group is not as large and leaves a hole in themiddle of the hydrophobic cluster.

FIGS. 9A-9H illustrate certain protein-aptamer interactions, asdescribed in Example 2. In FIG. 9A, Bn-dU1 occupies a pocket under asalt bridge at the homodimer interface. The UI base makes hydrogen bondsto the protein backbone at Val39 while the benzyl ring is sandwichedbetween the aliphatic side chain of Arg 56 and the disulfide bond ofCys43-Cys52. In FIG. 9B, Bn2 has an edgewise interaction with Trp40 andis nestled between the methylene side chains of Asn55 and Leu38. FIG. 9Cillustrates that the aromatic ring of Bn7 tucks up against the aliphaticportions of Asn54 and Asn55 side chains. FIG. 9D illustrates that theLeu38 and Ile75 side chains present a hydrophobic surface for the benzylring of Bn-dU8 to contact the protein. FIG. 9E illustrates that Bn16 hasedge-to-face π-stacking with Trp40 and van der Waals contact with thealiphatic region of Arg73. FIG. 9F illustrates that Pel7 is surroundedby the hydrophobic side chains of Leu38, Trp40, Arg73 and Ile75. Arg73makes a hydrogen bond to the amide linker of Pe-dU17 and a charge-chargeinteraction with the aptamer backbone. FIG. 9G illustrates Th18encircled by the hydrophobic side chains of Arg73, Ile75 and Phe84. FIG.9H illustrates that stacking interactions between the protein and theaptamer are present between Pro82 and Bn20 and U8, and Phe84 makesedge-to-face contact with U20. Bn20 makes additional hydrophobic contactto Ile77 and Lys80.

FIG. 10A shows the six co-crystal structures of traditional aptamers(PDB IDs: vWF, 3HXO; Thrombin, 3QLP; GlnRs tRNA, IEXD; Human IgG, 3AGV;MS2 coat protein, 6MSF; NF-kB, 1OOA) that were analyzed for the numberof polar contacts (hydrogen bonds plus charge-charge interactions), andthe contact surface area. The results are plotted versus the reportedbinding affinities for these six aptamer-target complexes (dark graybars) and for three SOMAmers (light gray bars) including the PDGF-SL5(4149-8_260) complex and two unpublished SOMAmer-target structures. Therelationship between the number of polar contacts and the contactsurface area for the six traditional aptamers was analyzed by linearregression, and the 99% confidence interval is indicated by the grayshading on the floor of the figure. FIG. 10B illustrates shapecomplementarity of the PDGF-SOMAmer complex as exhibited by Pe-dU17 andTh-dU18. Left, PDGF chain 1 is shown as a dark gray surface, Pe-dU17 andTh-dU18 are shown as space-filling representations. Right, same view asleft panel, except Pe-dU17 is shown as stick representation. FIG. 10Cillustrates detail of Bn-dU 1 interaction with PDGF, with PDGF chain 1shown as a dark gray surface, PDGF chain 2 shown as a light graysurface, and Bn-dU1 shown as a space-filling representation.

FIGS. 11A-11C show a comparison of SL5 and PDGFRβ binding to PDGF-BB.FIG. 11A receptor co-crystal showing PDGF homodimer (chain 1, mediumgray; chain 2, light gray) and receptor extracellular domain coloreddark gray (from Shim, A. H., et al. (2010) Proc. Natl. Acad. Sci. USA107(25): 11307). FIG. 11B complex of PDGF homodimer (chain 1, mediumgray; chain 2, light gray) and SL5 (dark gray). FIG. 11C shows the aminoacid sequence of the PDGF-B mature form. Box shading indicates contactresidues to 4149-8_260, PDGFRβ or both. PDGF residues that make a 4 Åcontact with 4149-8_260, but do not contact PDGFRβ are boxed with noshading. PDGF residues that make 4 Å contact with PDGFRβ, but do notcontact 4149-8_260 are boxed with dark gray shading. PDGF residues thatmake 4 Å contact with both 4149-8_260 and PDGFRβ are boxed with mediumgray shading.

FIG. 12 illustrates certain exemplary C-5 pyrimidine modifications thatmay be incorporated into aptamers, such as slow off-rate aptamers.

FIG. 13 shows a representative plot of the inhibition of PDGF-BB-inducedPDGF Rβ phosphorylation in Hs27 fibroblasts with SOMAmers 4149-8_379(labeled as OH-4149-8_379) or 5′ amino-linker modified SOMAmer4149-8_379 (labeled as N-4149-8_379), as described in Example 4.

FIG. 14 shows the consensus sequence for a set of PDGF-binding clonesfrom the SELEX pool as determined by 454 pyrosequencing and nucleotidefrequency at each position, as described in Example 5.

FIG. 15 shows K_(d) ratios for modified aptamers based on parent aptamer4867-31_143, in which a particular Nap-dU nucleobase has been replacedby another modified dU nucleobase, relative to the parent aptamer(numbers <1 indicate the modified aptamer has greater affinity than theparent aptamer, and numbers >1 indicate the modified aptamer has loweraffinity than the parent aptamer); as described in Example 7.

FIG. 16 shows the consensus sequence for a set of VEGF-binding clonesfrom the SELEX pool as determined by 454 pyrosequencing and nucleotidefrequency at each position, as described in Example 7.

FIG. 17 shows percent VEGFR2 phosphorylation in human umbilical veinendothelial cells (HUVECs) stimulated with VEGF-121 or VEGF-165 and VEGFaptamers 4867-31_43 and 4867-31_192, as described in Example 9.

FIG. 18A shows inhibition of PDGF-induced PDGF Rβ phosphorylation inHs27 fibroblasts with PDGF aptamer 4149-8_379 (open circles) andPDGF/VEGF aptamer construct 4149-8_401 (closed circles); and FIG. 18Bshows inhibition of VEGF-induced VEGF R2 phosphorylation in HUVECs withVEGF aptamer 4867-31_192 (open circles) and PDGF/VEGF aptamer construct4149-8_401 (closed circles); as described in Example 11.

FIGS. 19A-B show simultaneous binding of PDGF and VEGF by PDGF/VEGFaptamer construct SL012 (20 kDa PEG-N-4149-8_401) on microtiter platescoated with the VEGF with addition of biotinylated PDGF (FIG. 19A), andmicrotiter plates coated with PDGF with the addition of biotinylatedVEGF (FIG. 19B), as described in Example 12.

FIGS. 20A-B show simultaneous binding of PDGF and VEGF by PDGF/VEGFaptamer constructs SL1012 (20 kDa PEG-N-4149-8_401) (FIG. 20A) andSL1013 (40 kDA PEG-N-4149-8-401) (FIG. 20B), on microtiter plates thatwere coated with PDGF with the addition of biotinylated VEGF, asdescribed in Example 12.

FIGS. 21A-B show the simultaneous binding of PDGF and VEGF by variousPDGF/VEGF aptamer constructs on (FIG. 21A) microtiter plates coated withVEGF with the addition of biotinylated PDGF, and (FIG. 21B) microtiterplates coated with PDGF with the addition of biotinylated VEGF, asdescribed in Example 12.

FIG. 22A shows a plot of a number of polar contacts (defined as the sumof hydrogen bonds and charge-charge interactions) versus interface areafor traditional aptamers (diamonds) and SOMAmers (circles) (the linearregression fit has an R²=0.91 with a slope of 0.016; dashed linesrepresent the 99% confidence intervals of this trend, with the SOMAmersfalling outside those boundaries). FIG. 22B shows a plot of free energybinding versus polar contacts for traditional aptamers (diamonds) andSOMAmers (circles) (the linear regression fit has an R²=0.64 with aslope of 0.073), and FIG. 22C shows a table showing variousthermodynamic properties and contact characteristics of six previousaptamer-protein crystal structures and three SOMAmer-protein crystalstructures, including PDGF-BB: 4149-8_260, as described in Example 2.Interaction features for aptamers and SOMAmers bound to protein targets(FIG. 22C references: (a) Convery et al. (1998) Nat. Struct. Biol. 5(2):133; (b) Nomura et al. (2010) Nucleic Acids Res. 38(21): 7822; (c)Pagano et al. (2008) Biophys. J. 94(2):562; (d) Huang et al. (2003)Proc. Natl. Acad. Sci. USA 100(16):9268; (e) Huang et al. (2009)Structure 17(11):1476; (f) Bullock et al. (2000) Nat. Struct. Biol.7(6):497. Free energy calculations were determined from the measuredbinding affinities for SOMAmers, or the published K_(d) values using thefollowing temperatures: MS2, thrombin, NFkB, vWF, and GlnRs, roomtemperature (296 K); IgG, 298 K; SOMAmers, 310 K. SOMAmers show a trendtoward higher binding affinities; average free energy of binding, or −ΔGvalue, is 11.4±1.3 kcal/mol for the six aptamers and 14.3 kcal/mol±0.8kcal/mol for the three SOMAmers. Protein contact atoms within 4 Å ofeach ligand were determined in PyMOL. Interface area calculations weremade with PISA (aptamers) (Krissinel et al. (2007) J. Mol. Biol.372(3):774) or PyMOL (SOMAmers) (DeLano (2002) The Pymol MolecularGraphics System, Delano Scientific, San Carlos, Calif.). Within thisrelatively small data set of crystallographically evaluatedinteractions, aptamers engage their targets with an average ligandefficiency of 0.21±0.14 kcal/mol per nonhydrogen contact atom, comparedto 0.16±0.04 kcal/mol per non-hydrogen contact atom for SOMAmers. Freeenergies of binding per interface area are also similar, with an averagevalue of 0.017±0.009 kcal·mol⁻¹·Å⁻² for aptamers and 0.012±0.001kcal·mol⁻¹·Å⁻² for SOMAmers. The value of free energy of binding perpolar contact, calculated from values in the table, is about twice aslarge for SOMAmers (average of 1.75±0.36 kcal/mol per polar contact) asfor aptamers (0.89±0.56 kcal/mol per polar contact).

DETAILED DESCRIPTION

Reference will now be made in detail to representative embodiments ofthe invention. While the invention will be described in conjunction withthe enumerated embodiments, it will be understood that the invention isnot intended to be limited to those embodiments. On the contrary, theinvention is intended to cover all alternatives, modifications, andequivalents that may be included within the scope of the presentinvention as defined by the claims.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in and arewithin the scope of the practice of the present invention. The presentinvention is in no way limited to the methods and materials described.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art(s) to which this invention belongs. Although any methods,devices, and materials similar or equivalent to those described hereincan be used in the practice or testing of the invention, the preferredmethods, devices and materials are now described.

All publications, published patent documents, and patent applicationscited in this disclosure are indicative of the level of skill in theart(s) to which the disclosure pertains. All publications, publishedpatent documents, and patent applications cited herein are herebyincorporated by reference to the same extent as though each individualpublication, published patent document, or patent application wasspecifically and individually indicated as being incorporated byreference.

As used in this disclosure, including the appended claims, the singularforms “a,” “an,” and “the” include plural references, unless the contentclearly dictates otherwise, and are used interchangeably with “at leastone” and “one or more.” Thus, reference to “an aptamer” includesmixtures of aptamers, and the like.

As used herein, the term “about” represents an insignificantmodification or variation of the numerical value such that the basicfunction of the item to which the numerical value relates is unchanged.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “contains,” “containing,” and any variations thereof, areintended to cover a non-exclusive inclusion, such that a process,method, product-by-process, or composition of matter that comprises,includes, or contains an element or list of elements does not includeonly those elements but may include other elements not expressly listedor inherent to such process, method, product-by-process, or compositionof matter.

As used herein, the term “nucleotide” refers to a ribonucleotide or adeoxyribonucleotide, or a modified form thereof, as well as an analogthereof. Nucleotides include species that include purines (e.g.,adenine, hypoxanthine, guanine, and their derivatives and analogs) aswell as pyrimidines (e.g., cytosine, uracil, thymine, and theirderivatives and analogs).

As used herein, “nucleic acid,” “oligonucleotide,” and “polynucleotide”are used interchangeably to refer to a polymer of nucleotides andinclude DNA, RNA, DNA/RNA hybrids and modifications of these kinds ofnucleic acids, oligonucleotides and polynucleotides, wherein theattachment of various entities or moieties to the nucleotide units atany position are included. The terms “polynucleotide,”“oligonucleotide,” and “nucleic acid” include double- or single-strandedmolecules as well as triple-helical molecules. Nucleic acid,oligonucleotide, and polynucleotide are broader terms than the termaptamer and, thus, the terms nucleic acid, oligonucleotide, andpolynucleotide include polymers of nucleotides that are aptamers but theterms nucleic acid, oligonucleotide, and polynucleotide are not limitedto aptamers.

As used herein, the terms “modify”, “modified”, “modification”, and anyvariations thereof, when used in reference to an oligonucleotide, meansthat at least one of the four constituent nucleotide bases (i.e., A, G,T/U, and C) of the oligonucleotide is an analog or ester of a naturallyoccurring nucleotide. In some embodiments, the modified nucleotideconfers nuclease resistance to the oligonucleotide. In some embodiments,the modified nucleotides lead to predominantly hydrophobic interactionsof aptamers with protein targets resulting in high binding efficiencyand stable co-crystal complexes. A pyrimidine with a substitution at theC-5 position is an example of a modified nucleotide. Modifications caninclude backbone modifications, methylations, unusual base-pairingcombinations such as the isobases isocytidine and isoguanidine, and thelike. Modifications can also include 3′ and 5′ modifications, such ascapping. Other modifications can include substitution of one or more ofthe naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and those with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators, andthose with modified linkages (e.g., alpha anomeric nucleic acids, etc.).Further, any of the hydroxyl groups ordinarily present on the sugar of anucleotide may be replaced by a phosphonate group or a phosphate group;protected by standard protecting groups; or activated to prepareadditional linkages to additional nucleotides or to a solid support. The5′ and 3′ terminal OH groups can be phosphorylated or substituted withamines, organic capping group moieties of from about 1 to about 20carbon atoms, polyethylene glycol (PEG) polymers in some embodimentsranging from about 10 to about 80 kDa, PEG polymers in some embodimentsranging from about 20 to about 60 kDa, or other hydrophilic orhydrophobic biological or synthetic polymers. In some embodiments,modifications are of the C-5 position of pyrimidines. Thesemodifications can be produced through an amide linkage directly at theC-5 position or by other types of linkages.

Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclicsugar analogs, α-anomeric sugars, epimeric sugars such as arabinose,xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses,acyclic analogs and abasic nucleoside analogs such as methyl riboside.As noted above, one or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups includeembodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S(“dithioate”), (O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical. Substitution ofanalogous forms of sugars, purines, and pyrimidines can be advantageousin designing a final product, as can alternative backbone structureslike a polyamide backbone, for example.

As used herein, the term “nuclease” refers to an enzyme capable ofcleaving the phosphodiester bond between nucleotide subunits of anoligonucleotide. As used herein, the term “endonuclease” refers to anenzyme that cleaves phosphodiester bond(s) at a site internal to theoligonucleotide. As used herein, the term “exonuclease” refers to anenzyme which cleaves phosphodiester bond(s) linking the end nucleotidesof an oligonucleotide. Biological fluids typically contain a mixture ofboth endonucleases and exonucleases.

As used herein, the terms “nuclease resistant” and “nuclease resistance”refers to the reduced ability of an oligonucleotide to serve as asubstrate for an endo- or exonuclease, such that, when contacted withsuch an enzyme, the oligonucleotide is either not degraded or isdegraded more slowly than an oligonucleotide composed of unmodifiednucleotides.

As used herein, the term “C-5 modified pyrimidine” refers to apyrimidine with a modification at the C-5 position including, but notlimited to, those moieties illustrated in FIG. 12. Examples of a C-5modified pyrimidine include those described in U.S. Pat. Nos. 5,719,273and 5,945,527. Examples of a C-5 modification include substitution ofdeoxyuridine at the C-5 position with a substituent independentlyselected from: benzylcarboxyamide (alternatively benzylaminocarbonyl)(Bn), naphthylmethylcarboxyamide (alternativelynaphthylmethylaminocarbonyl) (Nap), tryptaminocarboxyamide(alternatively tryptaminocarbonyl) (Trp), phenethylcarboxyamide(alternatively phenethylamino carbonyl) (Pe),thiophenylmethylcarboxyamide (alternativelythiophenylmethylaminocarbonyl) (Th) and isobutylcarboxyamide(alternatively isobutylaminocarbonyl) (iBu) as illustrated immediatelybelow.

Chemical modifications of a C-5 modified pyrimidine can also be combinedwith, singly or in any combination, 2′-position sugar modifications,modifications at exocyclic amines, and substitution of 4-thiouridine andthe like.

Representative C-5 modified pyrimidines include:5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-benzylcarboxyamide)-2′-O-methyluridine,5-(N-benzylcarboxyamide)-2′-fluorouridine,5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-isobutylcarboxyamide)-2′-O-methyluridine,5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PedU),5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine (ThdU),5-(N-isobutylcarboxyamide)-2′-fluorouridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,5-(N-tryptaminocarboxyamide)-2′-fluorouridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU),5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine or5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine).

Nucleotides can be modified either before or after synthesis of anoligonucleotide. A sequence of nucleotides in an oligonucleotide may beinterrupted by one or more non-nucleotide components. A modifiedoligonucleotide may be further modified after polymerization, such as,for example, by conjugation with any suitable labeling component.

As used herein, the term “at least one pyrimidine,” when referring tomodifications of a nucleic acid, refers to one, several, or allpyrimidines in the nucleic acid, indicating that any or all occurrencesof any or all of C, T, or U in a nucleic acid may be modified or not.

As used herein, A, C, G, U and T denote dA, dC, dG, dU and dTrespectively, unless otherwise specified.

As used herein, “nucleic acid ligand,” “aptamer,” and “clone” are usedinterchangeably to refer to a non-naturally occurring nucleic acid thathas a desirable action on a target molecule. A desirable actionincludes, but is not limited to, binding of the target, catalyticallychanging the target, reacting with the target in a way that modifies oralters the target or the functional activity of the target, covalentlyattaching to the target (as in a suicide inhibitor), and facilitatingthe reaction between the target and another molecule. In someembodiments, the action is specific binding affinity for a targetmolecule, such target molecule being a three dimensional chemicalstructure other than a polynucleotide that binds to the nucleic acidligand through a mechanism which is independent of Watson/Crick basepairing or triple helix formation, wherein the aptamer is not a nucleicacid having the known physiological function of being bound by thetarget molecule. Aptamers to a given target include nucleic acids thatare identified from a candidate mixture of nucleic acids, where theaptamer is a ligand of the target, by a method comprising: (a)contacting the candidate mixture with the target, wherein nucleic acidshaving an increased affinity to the target relative to other nucleicacids in the candidate mixture can be partitioned from the remainder ofthe candidate mixture; (b) partitioning the increased affinity nucleicacids from the remainder of the candidate mixture; and (c) amplifyingthe increased affinity nucleic acids to yield a ligand-enriched mixtureof nucleic acids, whereby aptamers of the target molecule areidentified. It is recognized that affinity interactions are a matter ofdegree; however, in this context, the “specific binding affinity” of anaptamer for its target means that the aptamer binds to its targetgenerally with a much higher degree of affinity than it binds to other,non-target, components in a mixture or sample. An “aptamer” or “nucleicacid ligand” is a set of copies of one type or species of nucleic acidmolecule that has a particular nucleotide sequence. An aptamer caninclude any suitable number of nucleotides. “Aptamers” refer to morethan one such set of molecules. Different aptamers can have either thesame or different numbers of nucleotides. Aptamers may be DNA or RNA andmay be single stranded, double stranded, or contain double stranded ortriple stranded regions.

As used herein, a “SOMAmer” or Slow Off-Rate Modified Aptamer refers toan aptamer (including an aptamers comprising at least one nucleotidewith a hydrophobic modification) with an off-rate (t_(1/2)) of ≥30minutes, ≥60 minutes, ≥90 minutes, ≥120 minutes, ≥150 minutes, ≥180minutes, ≥210 minutes, or ≥240 minutes. In some embodiments, SOMAmersare generated using the improved SELEX methods described in U.S. Pat.No. 7,947,447, entitled “Method for Generating Aptamers with ImprovedOff-Rates”.

As used herein, “protein” is used synonymously with “peptide,”“polypeptide,” or “peptide fragment.” A “purified” polypeptide, protein,peptide, or peptide fragment is substantially free of cellular materialor other contaminating proteins from the cell, tissue, or cell-freesource from which the amino acid sequence is obtained, or substantiallyfree from chemical precursors or other chemicals when chemicallysynthesized.

As used herein, “co-crystal structure” or “co-crystal complex” is acrystal structure comprising two or more interacting molecules.

As used herein, “cardiovascular condition or disease” means a conditionor disease related to heart and its vascular system. Some examples ofsuch conditions or diseases are aneurysm, angina, arrhythmia,atherosclerosis, atrial fibrillation, congestive heart failure,cardiomyopathy, coronary heart disease, restenosis, ischemia, leftventricular hypertrophy, peripheral vascular disease, myocardialinfarction, hypertension, valvular heart disease and restrictive heartdisease.

As used herein, “fibrosis” means a disease or condition caused by theformation of an excessive and abnormal amount of fibrous connectivetissue in an organ resulting in thickening and scarring of connectivetissue, leading to malfunction of the organ. Examples of such diseasesand conditions are pulmonary fibrosis, renal fibrosis, liver fibrosisand cystic fibrosis.

As used herein, “AMD” or “age related macular degeneration” or “maculardegeneration” means a condition of the eye that is caused by damage tothe retina and results in a loss of vision in the center of the visualfield, called the macula. AMD occurs in “wet” and “dry” forms. In “wet”AMD, blood vessels grow from the choroid behind the retina. In “dry”AMD, cellular debris (drusen) accumulates between the retina andchoroid. In either form, the retina can become detached.

As used herein, “ophthalmic disease” or “ophthalmic condition” or“ocular disease” or “ocular condition” refers to any disease orcondition that affects or involves occular neovascularization disorders,such as macular degeneration (“wet” and “dry”), retinopathy ofprematurity, diabetic retinopathy, neovascular glaucoma, cornealneovascularization, proliferative diabetic retinopathy (the most severestage of diabetic retinopathy), uveitis (an inflammatory condition ofthe eye that often leads to macular edema), cystoid macular edemafollowing cataract surgery, myopic degeneration (a condition in which apatient with a high degree of nearsightedness develops choroidalneovascularization), inflammatory macular degeneration (a condition inwhich a patient with inflammation in the macular area due to infectionsor other causes, develops choroidal neovascularization), and irisneovascularization (a serious complication of diabetic retinopathy orretinal vein occlusion involving new blood vessel growth on the surfaceof the iris).

As used herein, “renal disease” or “renal condition” refers to anydisease or condition that affects or involves proliferative kidneydisorders such as glomerulonephritis, masangial proliferative renaldiseases, polycystic kidney disease, kidney cancers, acute kidneyfailure, nephropathy, amyloidosis, edema, fibrosis, glomerular diseases,renal infarction and nephritis.

As used herein “cancer” means a disease or condition involvingunregulated and abnormal cell growth. Some examples of common cancersare bladder cancer, lung cancer, breast cancer, melanoma, colon andrectal cancer, lymphoma, endometrial cancer, pancreatic cancer, livercancer, renal cancer, prostate cancer, leukemia and thyroid cancer.

As used herein, “modulate” means to alter, either by increasing ordecreasing, the level of a peptide or polypeptide, or to alter, eitherby increasing or decreasing, the stability or activity of a peptide or apolypeptide. The term “inhibit” means to decrease the level of a peptideor a polypeptide or to decrease the stability or activity of a peptideor a polypeptide. As described herein, the protein which is modulated orinhibited is PDGF.

As used herein, the term “bioactivity” indicates an effect on one ormore cellular or extracellular process (e.g., via binding, signaling,etc.) which can impact physiological or pathophysiological processes.

As used herein, the terms “platelet-derived growth factor” and “PDGF”refer to PDGF A, B, C, and D isoforms and their homo or heterodimers AA,BB, AB, CC and DD. In some instances, context will determine whichisoform and/or heterodimer of PDGF is meant. For example, in someembodiments, the PDGF aptamers described herein bind to the PDGF-Bisoform and homo- and heterodimers comprising that isoform, although theaptamers may be described as binding to PDGF. Specifically included inthe definition are naturally-occurring human PDGF AA, AB, and BBisoforms and variants. As used herein, PDGF includes all mammalianspecies of PDGF, including human, canine, feline, murine, primate,equine, and bovine. A nonlimiting exemplary human PDGF-B isoformprecursor has the sequence shown in Swiss-Prot Accession No. P01127.1.Nonlimiting exemplary human PDGF-B isoform mature proteins may have thesequence of amino acids 82 to 241 or 82 to 190 of Swiss-Prot AccessionNo. P01127.1 (referred to herein as amino acids 1 to 160 or 1 to 109 ofPDGF-B).

As used herein, “PDGF receptor” refers to a receptor that is bound byand activated by PDGF, such as PDGF receptor α and PDGF receptor β. PDGFreceptors include the receptors of any mammalian species, including, butnot limited to, human, canine, feline, murine, equine, primate, andbovine. A nonlimiting exemplary human PDGFRβ precursor has the sequenceshown in Swiss-Prot Accession No. P09619.1. A nonlimiting exemplaryhuman PDGFRβ mature protein has the sequence of amino acids 33 to 1106of Swiss-Prot Accession No. P09619.1. A nonlimiting exemplary humanPDGFRα precursor has the sequence shown in Swiss-Prot Accession No.P16234.1. A nonlimiting exemplary human PDGFRα mature protein has thesequence of amino acids 24 to 1089 of Swiss-Prot Accession No. P16234.1.

A “PDGF aptamer” is an aptamer that is capable of binding to andmodifying the activity of PDGF. In some embodiments, a PDGF aptamerinhibits the activity of PDGF in vitro. In some embodiments, a PDGFaptamer inhibits the activity of PDGF in vivo. A nonlimiting exemplaryactivity of PDGF is PDGF-mediated phosphorylation of the PDGF receptor,such as PDGF receptor α (PDGF Rα) or PDGF receptor β (PDGF Rβ).

In some embodiments, the “VEGF aptamer” as defined herein is a monomer,dimer or a multimer construct, optionally connected by a linker.

As used herein, the terms “vascular endothelial growth factor”, and“VEGF” refer to naturally-occurring VEGF, including isoforms andvariants, such as VEGF-121, VEGF-145, VEGF-165, VEGF-183, VEGF-189, andVEGF-206. As used herein, VEGF includes all mammalian species of VEGF,including human, canine, feline, murine, primate, equine, and bovine. Anonlimiting exemplary human VEGF percursor has the sequence shown inSwiss-Prot Accession No. P15692.2. VEGF-121 is described, e.g., in Teeet al. (2001) Biochem. J. 359:219; Bornes et al. (2004) J. Biol. Chem.279:18717.

As used herein, “VEGF receptor” refers to a receptor that is bound byand activated by VEGF, such as VEGFR-1 and VEGFR-2. VEGF receptorsinclude the receptors of any mammalian species, including, but notlimited to, human, canine, feline, murine, equine, primate, and bovine.A nonlimiting exemplary human VEGFR-1 precursor has the sequence shownin Swiss-Prot Accession No. P17948.2. A nonlimiting exemplary humanVEGFR-1 mature protein has the sequence of amino acids 27 to 1338 ofSwiss-Prot Accession No. P17948.2. A nonlimiting exemplary human VEGFR-2precursor has the sequence shown in Swiss-Prot Accession No. P35968.2. Anonlimiting exemplary human VEGFR-2 mature protein has the sequence ofamino acids 20 to 1356 of Swiss-Prot Accession No. P35968.2.

A “VEGF aptamer” is an aptamer that is capable of binding to andmodifying the activity of VEGF. In some embodiments, a VEGF aptamerinhibits the activity of VEGF in vitro. In some embodiments, a VEGFaptamer inhibits the activity of VEGF in vivo. Nonlimiting exemplaryactivities of VEGF include VEGF-mediated phosphorylation of the VEGFreceptor, such as VEGFR-1 or VEGFR-2. In some embodiments, a VEGFaptamer is provided that competes for binding to VEGF-121 with aptamer4867-31_183.

In some embodiments, the “VEGF aptamer” as defined herein is a monomer,dimer or a multimer construct, optionally connected by a linker.

The terms “PDGF/VEGF aptamer construct” and “VEGF/PDGF aptamerconstruct” are used interchangeably to refer to a construct comprising aPDGF aptamer and a VEGF aptamer. The order of the words “PDGF” and“VEGF” in “PDGF/VEGF aptamer construct” and “VEGF/PDGF aptamerconstruct” is not indicative of how the aptamers are linked, e.g., theorder does not indicate which aptamer is located at the 5′-most positionof an aptamer construct and which aptamer is located at the 3′-mostposition in the aptamer construct. In some embodiments, a PDGF/VEGFaptamer construct is capable of binding PDGF and VEGF simultaneously. Insome embodiments, a PDGF/VEGF aptamer construct is capable of bindingeach of PDGF and VEGF separately. In a PDGF/VEGF aptamer construct, thePDGF aptamer and the VEGF aptamer may be linked covalently ornon-covalently, e.g., through a binding pair such as streptavidin andbiotin. A PDGF/VEGF aptamer construct may comprise a linker between thePDGF aptamer and the VEGF aptamer.

As used herein, “disease or condition mediated by PDGF” refers todiseases or conditions in which PDGF activity may directly or indirectlylead to the disease or condition. Nonlimiting exemplary diseases orconditions mediated by PDGF include cardiovascular diseases such asatherosclerosis, restenosis, cardiac hypertrophy related conditions, andvascular disorders; ophthalmic diseases such as macular degeneration;fibrosis; and cancers.

As used herein, “disease or condition mediated by VEGF” refers todiseases or conditions in which VEGF activity may directly or indirectlylead to the disease or condition. Nonlimiting exemplary diseases orconditions mediated by VEGF include cardiovascular diseases, autoimmunediseases, inflammatory rheumatic diseases, ophthalmic diseases, andcancers at various stages in the disease process. Nonlimiting examplesof cardiovascular diseases are atherosclerosis, restenosis, cardiachypertrophy related conditions, and vascular disorders. Non-limitingexamples of ophthalmic diseases are retinitis, macular degeneration,choroiditis, retinopathy, edema, glaucoma, and cataract.

As used herein, the term “pharmaceutically acceptable” means approved bya regulatory agency of a federal or a state government or listed in theU.S. Pharmacopoeia or other generally recognized pharmacopoeia for usein animals and, more particularly, in humans. The term “carrier” refersto a diluent, adjuvant, excipient, or vehicle with which the therapeuticis administered and includes, but is not limited to, such sterileliquids as water and oils.

As used herein, the term “pharmaceutically acceptable salt” or “salt” ofa PDGF aptamer, VEGF aptamer or a PDGF/VEGF aptamer construct is aproduct of the disclosed compound that contains an ionic bond and istypically produced by reacting the disclosed compound with either anacid or a base, suitable for administering to an individual. Apharmaceutically acceptable salt can include, but is not limited to,acid addition salts including hydrochlorides, hydrobromides, phosphates,sulphates, hydrogen sulphates, alkylsulphonates, arylsulphonates,arylalkylsulfonates, acetates, benzoates, citrates, maleates, fumarates,succinates, lactates, and tartrates; alkali metal cations such as Li,Na, K, alkali earth metal salts such as Mg or Ca, or organic aminesalts.

As used herein, the term “pharmaceutical composition” is a formulationcomprising a PDGF aptamer, a VEGF aptamer, or a PDGF/VEGF aptamerconstruct in a form suitable for administration to an individual. Apharmaceutical composition is typically formulated to be compatible withits intended route of administration. Examples of routes ofadministration include, but are not limited to, oral and parenteral,e.g., intravenous, intradermal, subcutaneous, inhalation, topical,transdermal, transmucosal, and rectal administration.

As used herein, the term “therapeutically effective amount” generallymeans the amount necessary to ameliorate at least one symptom of adisorder or condition to be prevented, reduced, or treated as describedherein. The phrase “therapeutically effective amount” as it relates tothe PDGF aptamers, VEGF aptamers, or PDGF/VEGF aptamer constructs of thepresent disclosure means the aptamer dosage that provides the specificpharmacological response for which the aptamer is administered in asignificant number of individuals in need of such treatment. It isemphasized that a therapeutically effective amount of an aptamer that isadministered to a particular individual in a particular instance willnot always be effective in treating the conditions/diseases describedherein, even though such dosage is deemed to be a therapeuticallyeffective amount by those of skill in the art.

The terms “SELEX” and “SELEX process” are used interchangeably herein torefer generally to a combination of (1) the selection of nucleic acidsthat interact with a target molecule in a desirable manner, for exampleby binding with high affinity to a protein, with (2) the amplificationof those selected nucleic acids. The SELEX process can be used toidentify aptamers with high affinity to a specific target molecule.

SELEX generally includes preparing a candidate mixture of nucleic acids,binding of the candidate mixture to the desired target molecule to forman affinity complex, separating the affinity complexes from the unboundcandidate nucleic acids, separating and isolating the nucleic acid fromthe affinity complex, purifying the nucleic acid, and identifying aspecific aptamer sequence. The process may include multiple rounds tofurther refine the affinity of the selected aptamer. The process caninclude amplification steps at one or more points in the process. See,e.g., U.S. Pat. No. 5,475,096, entitled “Nucleic Acid Ligands.” TheSELEX process can be used to generate an aptamer that covalently bindsits target as well as an aptamer that non-covalently binds its target.See, e.g., U.S. Pat. No. 5,705,337 entitled “Systematic Evolution ofNucleic Acid Ligands by Exponential Enrichment: Chemi-SELEX.”

The SELEX process can be used to identify high-affinity aptamerscontaining modified nucleotides that confer improved characteristics onthe aptamer, such as, for example, improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX process-identified aptamers containing modifiednucleotides are described in U.S. Pat. No. 5,660,985, entitled “HighAffinity Nucleic Acid Ligands Containing Modified Nucleotides,” whichdescribes oligonucleotides containing nucleotide derivatives chemicallymodified at the C5 and/or 2′-positions of pyrimidines. U.S. Pat. No.5,580,737, see supra, describes highly specific aptamers containing oneor more nucleotides modified with 2′-amino (2′-NH₂), 2′-fluoro (2′-F),and/or 2′-O-methyl (2′-OMe). See also, U.S. Patent Publication No.20090098549, entitled “SELEX and PHOTOSELEX,” which describes nucleicacid libraries having expanded physical and chemical properties andtheir use in SELEX and photoSELEX.

SELEX can also be used to identify aptamers that have desirable off-ratecharacteristics. See U.S. Pat. No. 7,947,447, entitled “Method forGenerating Aptamers with Improved Off-Rates,” which describes improvedSELEX methods for generating aptamers that can bind to target molecules.Methods for producing aptamers and photoaptamers having slower rates ofdissociation from their respective target molecules are described. Themethods involve contacting the candidate mixture with the targetmolecule, allowing the formation of nucleic acid-target complexes tooccur, and performing a slow off-rate enrichment process wherein nucleicacid-target complexes with fast dissociation rates dissociate and do notreform, while complexes with slow dissociation rates remain intact.Additionally, the methods include the use of modified nucleotides in theproduction of candidate nucleic acid mixtures to generate aptamers withimproved off-rate performance (see U.S. Patent Publication No.2009/0098549, entitled “SELEX and PhotoSELEX”). (See also U.S. Pat. No.7,855,054 and U.S. Patent Publication No. 2007/0166740). Each of theseapplications is incorporated herein by reference in its entirety.

In some embodiments, methods of selecting aptamers that bind to a targetmolecule are provided, comprising: (a) preparing a candidate mixture ofnucleic acids, wherein the candidate mixture comprises modified nucleicacids in which at least one pyrimidine in at least one, or in each,nucleic acid of the candidate mixture is chemically modified at theC5-position; (b) contacting the candidate mixture with a targetmolecule, wherein nucleic acids having an increased affinity to thetarget molecule relative to other nucleic acids in the candidate mixturebind the target molecule, forming nucleic acid-target moleculecomplexes; (c) partitioning the increased affinity nucleic acids fromthe remainder of the candidate mixture; and (d) amplifying the increasedaffinity nucleic acids to yield a mixture of nucleic acids enriched innucleic acid sequences that are capable of binding to the targetmolecule with increased affinity, whereby an aptamer to the targetmolecule is identified. In certain embodiments, the method furtherincludes performing a slow off-rate enrichment process.

“Target” or “target molecule” or “target” refers herein to any compoundupon which a nucleic acid can act in a desirable manner. A targetmolecule can be a protein, peptide, nucleic acid, carbohydrate, lipid,polysaccharide, glycoprotein, hormone, receptor, antigen, antibody,virus, pathogen, toxic substance, substrate, metabolite, transitionstate analog, cofactor, inhibitor, drug, dye, nutrient, growth factor,cell, tissue, any portion or fragment of any of the foregoing, etc.,without limitation. Virtually any chemical or biological effector may bea suitable target. Molecules of any size can serve as targets. A targetcan also be modified in certain ways to enhance the likelihood orstrength of an interaction between the target and the nucleic acid. Atarget can also include any minor variation of a particular compound ormolecule, such as, in the case of a protein, for example, minorvariations in amino acid sequence, disulfide bond formation,glycosylation, lipidation, acetylation, phosphorylation, or any othermanipulation or modification, such as conjugation with a labelingcomponent, which does not substantially alter the identity of themolecule. A “target molecule” or “target” is a set of copies of one typeor species of molecule or multimolecular structure that is capable ofbinding to an aptamer. “Target molecules” or “targets” refer to morethan one such set of molecules. Embodiments of the SELEX process inwhich the target is a peptide are described in U.S. Pat. No. 6,376,190,entitled “Modified SELEX Processes Without Purified Protein.”

Exemplary PDGF Aptamers

The PDGF aptamers of the instant disclosure were identified using theimproved SELEX method for identifying aptamers having slow off-rates asdescribed in Example 1, which describes a representative method for theselection and production of an aptamer that binds PDGF with a slowdissociation rate. A random DNA library composed of benzyl-dU (Bn-dU),dA, dC and dG was used for the selection. Using this method, the DNAaptamer to PDGF-BB designated as aptamer 4149-8_1 (SEQ ID NO: 1) wasidentified.

Using aptamer 4149-8_1 (SEQ ID NO: 1), studies were conducted toidentify the minimum sequence length required to maintain strongaffinity for PDGF. Systematic truncation from the 5′ and 3′ ends led toidentification of a core motif consisting of 29 nucleotides (4149-8_38;SEQ ID NO. 38). Aptamer 4149-8_38 exhibited high affinity binding toPDGF-BB (K_(d) value of 20 pM; FIG. 6).

Additional sequencing studies were conducted on the sequence pool fromwhich aptamer 4149-8_1 (SEQ ID NO: 1) was selected. 454 sequencing,which is a large-scale, high throughput method that uses parallelpyrosequencing, provides unbiased sample preparation and very accuratesequence analysis. The sequencing data was used to identify a consensussequence for a PDGF aptamer as shown in FIG. 3. Furthermore, nucleotidesubstitution studies illustrated in FIG. 6 led to the discovery that sixof eight BndU positions in the consensus sequence were desirable forPDGF binding, but four BndU positions could be replaced with dT withlittle or no loss of binding activity. A consensus sequence is shown inFIG. 3A, along with a graphic representation of the nucleotide frequencyat each position relative to the apatmer 4149-8_1 (SEQ ID NO: 1).

In some embodiments, a PDGF aptamer comprises the sequence:

(SEQ ID NO: 500) 5′-NZVSL_(n)S′V′ZACNN_(m)GCGZZZAZAGCG-3′,whereinV is selected from an A, C or G;V′ is selected from a C, G or Z, wherein V′ is complementary to V;S and S′ are independently selected from a C or G, wherein S and S′ arecomplementary to each other;each N is independently selected from any naturally occurring ormodified nucleotide;each Z is independently selected from a modified pyrimidine;L is selected from any naturally occurring or modified nucleotide, ahydrocarbon linker, a polyethylene glycol linker or a combinationthereof;n is 0 to 20; and m is 0 to 20; and wherein one or more nucleotideinsertions are optionally included.

In some embodiments, a PDGF aptamer comprises the sequence:

(SEQ ID NO: 501) 5′-ZZVSL_(n)S′V′ZACNN_(m)GCGZZZAZAGCG-3′,wherein V, V′, N, S, S′, Z, L, n, and m are as defined above.

In some embodiments, a PDGF aptamer comprises the sequence:

(SEQ ID NO: 502) 5′-ZZVCL_(n)GV′ZACNMGCGZZZAZAGCG-3′,wherein Z, V, V′, N, Z, L, and n are as defined above and M is selectedfrom C and A.

In some embodiments, a PDGF aptamer comprises the sequence:

(SEQ ID NO: 503) 5′-ZZACL_(n)GZZACACGCGZZZAZAGCG-3′,wherein Z, L, and n are as defined above.

In some embodiments, a PDGF aptamer comprises the sequence:

(SEQ ID NO: 504) 5′-ZZACGACZACGZZACACGCGZZZAZAGCG-3′,wherein Z is as defined above.

In some embodiments, a PDGF aptamer comprises the sequence:

(SEQ ID NO: 507) 5-ZVSL_(n)S′V′ZACNN_(m)GCGZZZAZAG-3′,whereinV is selected from an A, C or G;V′ is selected from a C, G or Z, wherein V′ is complementary to V;S and S′ are independently selected from a C or G, wherein S and S′ arecomplementary to other;each N is independently selected from modified or unmodified nucleotide;each Z is independently selected from a modified pyrimidine;L is selected from a substituted or unsubstituted C₂-C₂₀ linker and amodified or unmodified nucleotide;n is 1 to 50; and m is 0 to 50; andwherein one or more nucleotide insertions are optionally included.

In some embodiments, a PDGF aptamer comprises the sequence:

(SEQ ID NO: 508) 5′-Z′ZVSL_(n)S′V′ZACNN_(m)GCGZZZAZAGC-3′,wherein Z′ is a modified pyrimidine or dT; and V, V′, N, S, S′, Z, L, n,and m are as defined above.

In some embodiments, a PDGF aptamer comprises the sequence:

(SEQ ID NO: 509) 5′-Z′ZVCL_(n)GV′ZACNMGCGZZZAZAGC-3′,wherein Z, Z′, V, V′, N, Z, L, and n are as defined above and M isselected from C and A.

In some embodiments, a PDGF aptamer comprises the sequence:

(SEQ ID NO: 510) 5′-Z′ZACL_(n)GZZACACGCGZZZAZAGC-3′,wherein Z, Z′, L, and n are as defined above.

In some embodiments, a PDGF aptamer comprises the sequence:

(SEQ ID NO: 511) 5′-Z′ZACGACZACGZZACACGCGZZZAZAGC-3′,wherein Z and Z′ are as defined above.

In some embodiments, a PDGF aptamer comprises the sequence:

(SEQ ID NO: 505) 5′-ZABL_(p)GYZABK_(q)GCGZZYDYAG-3′wherein each Z is, independently, a modified pyrimidine;each B is independently selected from C and a substituted orunsubstituted C₂-C₁₀ linker;each L is independently selected from a substituted or unsubstitutedC₂-C₁₀ linker, a hexaethylene glycol linker, and a modified orunmodified nucleotide, wherein p is 1 to 10;each Y is independently selected from a modified or unmodifiedpyrimidine;each K is independently selected from a substituted or unsubstitutedC₂-C₁₀ linker, a hexaethylene glycol linker, and a modified orunmodified nucleotide, wherein q is 1 to 5; andD is selected from A and a substituted or unsubstituted C₂-C₁₀ linker.

In some embodiments, a PDGF aptamer comprises the sequence:

(SEQ ID NO: 506) 5′-XZABL_(n)GYZABL_(n)GCGZZYDYAGBE-3′,wherein X is selected from a modified or unmodified pyrimidine and asubstituted or unsubstituted C₂-C₁₀ linker, or is absent; and E isselected from G and a substituted or unsubstituted C₂-C₁₀ linker, or isabsent.

An aptamer construct comprising the sequences NZVS (SEQ ID NO 761) and

(SEQ ID NO: 762) S′V′ZACNN_(m)GCGZZZAZAGCG,whereinV is selected from an A, C or G; V is selected from a C, G or Z, whereinV′ is selected from a C, G or Z, wherein V′ is complementary to V;S and S′ are independently selected from a C or G, wherein S and S′ arecomplementary to each other;N is independently selected from any naturally occurring or modifiednucleotide;Z is independently selected from a modified pyrimidine;m is 1 to 20; andwherein one or more nucleotide insertions are optionally included.

In some embodiments, Z is a modified uridine. In some embodiments, eachZ is independently selected from the C-5 modified pyrimidines as definedherein. In some embodiments, each Z is independently selected from

-   5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),-   5-(N-benzylcarboxyamide)-2′-O-methyluridine,-   5-(N-benzylcarboxyamide)-2′-fluorouridine,-   5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU),-   5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine (ThdU),-   5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),-   5-(N-tyrosylcarboxyamide)-2′-deoxyuridine (TyrdU),-   5-(N-3,4-methylenedioxybenzylcarboxyamide)-2′-deoxyuridine (MBndU),-   5-(N-4-fluorobenzylcarboxyamide)-2′-deoxyuridine (FBndU),-   5-(N-3-phenylpropylcarboxyamide)-2′-deoxyuridine (PPdU),-   5-(N-imidizolylethylcarboxyamide)-2′-deoxyuridine (ImdU),-   5-(N-isobutylcarboxyamide)-2′-O-methyluridine,-   5-(N-isobutylcarboxyamide)-2′-fluorouridine,-   5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),-   5-(N—R-threoninylcarboxyamide)-2′-deoxyuridine (ThrdU),-   5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,-   5-(N-tryptaminocarboxyamide)-2′-fluorouridine,-   5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridine    chloride,-   5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU),-   5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,-   5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine,-   5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine),-   5-(N-2-naphthylmethylcarboxyamide)-2′-deoxyuridine (2NapdU),-   5-(N-2-naphthylmethylcarboxyamide)-2′-O-methyluridine,-   5-(N-2-naphthylmethylcarboxyamide)-2′-fluorouridine,-   5-(N-1-naphthylethylcarboxyamide)-2′-deoxyuridine (NEdU),-   5-(N-1-naphthylethylcarboxyamide)-2′-O-methyluridine,-   5-(N-1-naphthylethylcarboxyamide)-2′-fluorouridine,-   5-(N-2-naphthylethylcarboxyamide)-2′-deoxyuridine (2NEdU),-   5-(N-2-naphthylethylcarboxyamide)-2′-O-methyluridine,-   5-(N-2-naphthylethylcarboxyamide)-2′-fluorouridine,-   5-(N-3-benzofuranylethylcarboxyamide)-2′-deoxyuridine (BFdU),-   5-(N-3-benzofuranylethylcarboxyamide)-2′-O-methyluridine,-   5-(N-3-benzofuranylethylcarboxyamide)-2′-fluorouridine,-   5-(N-3-benzothiophenylethylcarboxyamide)-2′-deoxyuridine (BTdU),-   5-(N-3-benzothiophenylethylcarboxyamide)-2′-O-methyluridine, and-   5-(N-3-benzothiophenylethylcarboxyamide)-2′-fluorouridine.

In certain embodiments, portions of the PDGF and/or VEGF aptamer (Y) maynot be necessary to maintain binding and certain portions of thecontiguous PDGF and/or VEGF aptamer can be modified, including, but notlimited to, replacement with a spacer or linker moiety. In theseembodiments, for example, Y can be represented as Y′-Q-Y″-Q′-Y′″,wherein Y′, Y″ and Y′″ are parts of a PDGF and/or VEGF aptamer orsegments of different PDGF and/or VEGF aptamers and Q and/or Q′ arespacers or linker molecules that modify certain nucleic acid features ofthe original PDGF and/or VEGF aptamer. When Q and Q′ are not present,Y′, Y″, and Y′″ represent one contiguous PDGF and/or VEGF aptamer (Y).

As used herein a “linker” is a molecular entity that connects two ormore molecular entities through covalent bond or non-covalentinteractions and can allow spatial separation of the molecular entitiesin a manner that preserves the functional properties of one or more ofthe molecular entities. A linker can also be known as a spacer.Appropriate linker sequences will be readily ascertained by those ofskill in the art based upon the present disclosure.

As used herein, a linker can comprise one or more molecules orsub-components, selected from the group including, but not limited to, apolynucleotide, a polypeptide, a peptide nucleic acid, a locked nucleicacid, an oligosaccharide, a polysaccharide, an antibody, an affybody, anantibody mimic, an aliphatic, aromatic or heteroaromatic carbonmolecule, a polyethylene glycol (PEG) molecule, a cell receptor, aligand, a lipid, any fragment or derivative of these structures, anycombination of the foregoing, or any other chemical structure orcomponent.

In some embodiments, at least one L is a polyethylene glycol linker. Insome embodiments, at least one L is a hexaethylene glycol linker. Insome embodiments, L is a substituted or unsubstituted C₂-C₁₀ linker. Insome embodiments, p is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, pis 1, 2, or 3. In some embodiments, at least one K is a polyethyleneglycol linker. In some embodiments, at least one K is a hexaethyleneglycol linker. In some embodiments, K is a substituted or unsubstitutedC₂-C₁₀ linker. In some embodiments, q is 1 or 2. In some embodiments, qis 1.

In various embodiments, m may be 0 to 20, 0 to 19, 0 to 18, 0 to 17, 0to 16, 0 to 15, 0 to 15, 0 to 14, 0 to 13, 0 to 12, 0 to 11, 0 to 10, 0to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, or 0 to 3.

In some embodiments, L may be a linker such as an 18-atom hexaethyleneglycol linker. In some embodiments, the L may be a combination ofnucleotides and a linker. As a nonlimiting example, the followingaptamers (SEQ ID NOs 67 and 69) include a hexaethylene glycol (Heg)linker:

(SEQ ID NO. 67) 5′-Bn-Bn-A-C-Heg-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn-A-Bn-A-G-C-G-3′ (SEQ ID NO. 69)5′-Bn-Bn-A-C-G-Heg-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- Bn-A-Bn-A-G-C-G-3′

wherein Bn is benzyl-dU and Heg is a hexaethylene glycol linker.

In some embodiments, an N may be replaced by a linker, such as in thefollowing aptamers:

(SEQ ID NO. 329) 5′-Bn-Bn-A-C-Heg-G-Bn-Bn-A-C-C3-G-C-G-Bn-Bn-Bn-A-Bn-A-G-C-3′ (SEQ ID NO. 408)5′-Bn-Bn-A-C-Heg-G-Bn-Bn-A-C-C3-C-G-Bn-Bn-Bn-A-Bn- A-G-3′wherein Bn is benzyl-dU, Heg is a hexaethylene glycol linker and C3 is athree carbon linker.

Further PDGF aptamers were identified using the improved SELEX methodfor identifying aptamers having slow off-rates as described in Example5, which describes a representative method for the selection andproduction of an aptamer that binds PDGF with a slow dissociation rate.A random DNA library composed of napthyl-dU (Nap-dU), dA, dC and dG wasused for the selection. PDGF aptamer 5169-4_26 was identified in thescreen.

In some embodiments, an aptamer that specifically binds PDGF isprovided, wherein the aptamer competes for binding to PDGF with PDGFaptamer 5169-4_26. In some such embodiments, the aptamer comprises atleast one modified nucleoside comprising a hydrophobic nucleobasemodification. Further, in some such embodiments, the hydrophobicnucleobase modification is a modified pyrimidine. In some embodiments,each modified pyrimidine may be independently selected from5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-benzylcarboxyamide)-2′-O-methyluridine,5-(N-benzylcarboxyamide)-2′-fluorouridine,5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PedU),5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine (ThdU),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-isobutylcarboxyamide)-2′-O-methyluridine,5-(N-isobutylcarboxyamide)-2′-fluorouridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,5-(N-tryptaminocarboxyamide)-2′-fluorouridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU),5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine, and5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine).

In some embodiments, an aptamer that specifically bind PDGF is provided,wherein the aptamer comprises the sequence:

(SEQ ID NO. 512) 5′-ACAL_(n)ZGZAZGL_(m)ZLZ-3′;wherein each Z is, independently, a modified pyrimidine; each L isindependently selected from a substituted or unsubstituted C₂-C₅₀linker, a polyethylene glycol linker, and a modified or unmodifiednucleotide; n is 1 to 5; and m is 1 to 10.

In some embodiments, each Z is independently selected from5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-benzylcarboxyamide)-2′-O-methyluridine,5-(N-benzylcarboxyamide)-2′-fluorouridine,5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PedU),5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine (ThdU),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-isobutylcarboxyamide)-2′-O-methyluridine,5-(N-isobutylcarboxyamide)-2′-fluorouridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,5-(N-tryptaminocarboxyamide)-2′-fluorouridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU),5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine, and5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine). In someembodiments, at least one, at least two, at least three, at least four,or each Z is 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU).In some embodiments, n is 1, 2, 3, 4, or 5. In some embodiments, m is 1,2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, n is 3. In someembodiments, m is 4. In some such embodiments, each L is independentlyselected from a modified nucleotide, an unmodified nucleotide, and a C3linker.

A C₂-C₅₀ linker or spacer may be a backbone comprising a chain of 2 to50 carbon atoms (C₂-C₅₀) (saturated, unsaturated, straight chain,branched or cyclic), 0 to 10 aryl groups, 0 to 10 heteroaryl groups, and0 to 10 heterocyclic groups, optionally comprising an ether (—O—)linkage, (e.g., one or more alkylene glycol units, including but notlimited to one or more ethylene glycol units —O—(CH₂CH₂O)—; one or more1,3-propane diol units —O—(CH₂CH₂CH₂O)—, etc.); an amine (—NH—) linkage;an amide (—NC(O)—) linkage; and a thioether (—S—) linkage; etc.; whereineach backbone carbon atom may be independently unsubstituted (i.e.,comprising —H substituents) or may be substituted with one or moregroups selected from a C₁ to C₃ alkyl, —OH, —NH₂, —SH, —O—(C₁ to C₆alkyl), —S—(C₁ to C₆ alkyl), halogen, —OC(O)(C₁ to C₆ alkyl), —NH—(C₁ toC₆ alkyl), and the like. In some embodiments, a C₂-C₅₀ linker is aC₂-C₂₀ linker, a C₂-C₁₀ linker, a C₂-C₈ linker, a C₂-C₆ linker, a C₂-C₅linker, a C₂-C₄ linker, or a C₃ linker, wherein each carbon may beindependently substituted as described above.

In some embodiments, one or more nucleosides of a PDGF aptamer comprisea modification selected from a 2′-position sugar modification (such as a2′-amino (2′-NH₂), a 2′-fluoro (2′-F), or a 2′-O-methyl (2′-OMe)), amodification at a cytosine exocyclic amine, an internucleoside linkagemodification, and a 5-methyl-cytosine. In some embodiments, a PDGFaptamer comprises a 3′ cap, a 5′ cap, and/or an inverted deoxythymidineat the 3′ terminus.

In some embodiments, a PDGF aptamer comprises at least one modifiedinternucleoside linkage. In some embodiments, at least one, at leasttwo, at least three, at least four, or at least five internucleosidelinkages are phosphorothioate linkages.

In some embodiments, a PDGF aptamer has a sequence selected from thesequences shown in Tables 1, 2 and 6 to 9 (SEQ ID NOS: 1 to 499 and 517to 545). In some embodiments, a PDGF aptamer has a sequence selectedfrom the sequences shown in Table 1 and the sequences shown in Tables 6to 9 that bind PDGF with an affinity (K_(d)) of less than 10 nM. In someembodiments, a PDGF aptamer has a sequence that is at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% identicalto the sequences shown in Tables 1, 2, and 6 to 9 (SEQ ID NOS: 1 to 1 to499 and 517 to 545). In some embodiments, a PDGF aptamer has a sequencethat is at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% identical to the sequences shown in Table 1 and thesequences shown in Tables 6 to 9 that bind PDGF with an affinity (K_(d))of less than 10 nM.

The terms “sequence identity”, “percent sequence identity”, “percentidentity”, “% identical”, “% identity”, and variations thereof, whenused in the context of two nucleic acid sequences, are usedinterchangeably to refer to the number of nucleotide bases that are thesame in a query nucleic acid or a portion of a query nucleic acid, whenit is compared and aligned for maximum correspondence to a referencenucleic acid, divided by either (1) the number of nucleotide bases inthe query sequence between and including the most 5′ corresponding(i.e., aligned) nucleotide base and the most 3′ corresponding (i.e.,aligned) nucleotide base, or (2) the total length of the referencesequence, whichever is greater. Exemplary alignment of sequences forcomparison can be conducted, e.g., by the local homology algorithm ofSmith and Waterman (1981) Adv. Appl. Math. 2:482, by the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443,by the search for similarity method of Pearson and Lipman (1988) Proc.Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by visual inspection (see generally, Ausubel, F. M. et al.(1987), Current Protocols in Molecular Biology, Greene Publishing Assoc.and Wiley-Interscience).

One example of an algorithm that is suitable for determining percentsequence identity is the algorithm used in the basic local alignmentsearch tool (hereinafter “BLAST”), see, e.g. Altschul et al. (1990) J.Mol. Biol. 215:403 and Altschul et al. (1997) Nucleic Acids Res.15:3389. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information (hereinafter“NCBI”). The default parameters used in determining sequence identityusing the software available from NCBI, e.g., BLASTN (for nucleotidesequences) are described in McGinnis et al. (2004) Nucleic Acids Res.32:W20.

As used herein, when describing the percent identity of a nucleic acid,such as a PDGF aptamer, the sequence of which is at least, for example,about 95% identical to a reference nucleotide sequence, it is intendedthat the nucleic acid sequence is identical to the reference sequenceexcept that the nucleic acid sequence may include up to five pointmutations per each 100 nucleotides of the reference nucleic acidsequence. In other words, to obtain a desired nucleic acid sequence, thesequence of which is at least about 95% identical to a reference nucleicacid sequence, up to 5% of the nucleotides in the reference sequence maybe deleted or substituted with another nucleotide, or some number ofnucleotides up to 5% of the total number of nucleotides in the referencesequence may be inserted into the reference sequence (referred to hereinas an insertion). These mutations of the reference sequence to generatethe desired sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. Further, it is intended that a nucleotide base isconsidered “identical” for the purposes of determining percent identity,when the nucleotide base (1) is the same as the nucleotide base in thereference sequence, or (2) is derived from the nucleotide base in thereference sequence, or (3) is derived from the same nucleotide base fromwhich the nucleotide base in the reference sequence is derived. Forexample, 5-methyl cytosine is considered to be “identical” to cytosinefor the purposes of calculating percent identity. Similarly, themodified uridines shown in FIG. 12 are considered to be identical to oneanother for the purpose of determining percent identity. The referencesequence may be any one of the nucleotide sequences shown in SEQ ID NOS:1 to 437.

In some embodiments, the present disclosure provides a PDGF aptamerthat, upon binding PDGF, modulates a PDGF function. In some embodiments,a PDGF aptamer described herein inhibits PDGF-mediated phosphorylationof a PDGF receptor, such as PDGF Rα or PDGF Rβ. In some embodiments, aPDGF aptamer described herein inhibits PDGF-mediated phosphorylation ofPDGF Rβ. In various embodiments, the PDGF aptamer modulates a PDGFfunction in vivo, such as inhibiting PDGF-mediated receptorphosphorylation in vivo. In various embodiments, the PDGF aptamer has asequence selected from the sequences of SEQ ID NOS: 1 to 437. In variousembodiments, the PDGF aptamer is selected from the aptamers shown inTables 1 and 2. In various embodiments, the PDGF aptamer is selectedfrom the aptamers shown in Table 1. In some embodiments, the PDGFaptamer comprises at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 21, at least 22, at least 23, at least 24, at least 25, atleast 26, at least 27, at least 28, at least 29, or at least 30contiguous nucleotides of a sequence selected from SEQ ID NOS: 1 to 1 to499 and 517 to 545. In some embodiments, a PDGF aptamer consists of atleast 12, at least 13, at least 14, at least 15, at least 16, at least17, at least 18, at least 19, at least 20, at least 21, at least 21, atleast 22, at least 23, at least 24, at least 25, at least 26, at least27, at least 28, at least 29, or at least 30 contiguous nucleotides thatare identical in nucleobase sequence to a sequence selected from SEQ IDNOS: 1 to 1 to 499 and 517 to 545. In some embodiments, the PDGF aptamercomprises at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, at least 20, at least 21, atleast 21, at least 22, at least 23, at least 24, at least 25, at least26, at least 27, at least 28, at least 29, or at least 30 contiguousnucleotides of an aptamer shown in Table 1, 2, 6, 7, 8 or 9. In someembodiments, the PDGF aptamer comprises at least 12, at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least19, at least 20, at least 21, at least 21, at least 22, at least 23, atleast 24, at least 25, at least 26, at least 27, at least 28, at least29, or at least 30 contiguous nucleotides of an aptamer shown in Table 1or an aptamer shown in one of Tables 6 to 9 that binds PDGF with anaffinity (K_(d)) of less than 10 nM. In some embodiments, a PDGF aptamerconsists of at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, at least 20, at least 21, atleast 21, at least 22, at least 23, at least 24, at least 25, at least26, at least 27, at least 28, at least 29, or at least 30 contiguousnucleotides of an aptamer shown in Table 1, 2, 6, 7, 8 or 9. In someembodiments, a PDGF aptamer consists of at least 12, at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least19, at least 20, at least 21, at least 21, at least 22, at least 23, atleast 24, at least 25, at least 26, at least 27, at least 28, at least29, or at least 30 contiguous nucleotides of an aptamer shown in Table 1or an aptamer shown in one of Tables 6 to 9 that binds PDGF with anaffinity (K_(d)) of less than 10 nM.

In some embodiments, a PDGF aptamer has a nucleobase sequence selectedfrom the sequences of SEQ ID NOS. 500 to 512; 761 and 762. In someembodiments, a PDGF aptamer has the sequence of any one of SEQ ID NOS: 1to 1 to 499 and 517 to 545. In some embodiments, a PDGF aptamer is atleast 95% identical, at least 90% identical, at least 85% identical, atleast 80% identical, or at least 75% identical to any one of SEQ ID NOS:1 to 1 to 499 and 517 to 545. In any of the embodiments herein, a PDGFaptamer may comprise additional nucleotides or other chemical moietieson the 5′ end, the 3′ end, or both the 5′ and the 3′ end of the aptamer.

The PDGF aptamer can contain any number of nucleotides in addition tothe PDGF binding region. In various embodiments, the PDGF aptamer caninclude up to about 100 nucleotides, up to about 95 nucleotides, up toabout 90 nucleotides, up to about 85 nucleotides, up to about 80nucleotides, up to about 75 nucleotides, up to about 70 nucleotides, upto about 65 nucleotides, up to about 60 nucleotides, up to about 55nucleotides, up to about 50 nucleotides, up to about 45 nucleotides, upto about 40 nucleotides, up to about 35 nucleotides, up to about 30nucleotides, up to about 25 nucleotides, or up to about 20 nucleotides.

In some embodiments, the PDGF aptamer is selected from an aptamer thathas similar binding characteristics and ability to treat PDGF associatedatherosclerosis, macular degeneration, fibrosis, or cancer conditions asan aptamer selected from SEQ ID NOS: 1 to 1 to 499 and 517 to 545. Insome embodiments, a PDGF aptamer binds to the same region of a PDGF-Bmonomer (in the context of a PDGF-BB or PDGF-AB dimer) as an aptamerselected from the aptamers shown in Tables 1, 2, and 6 to 9. In someembodiments, a PDGF aptamer binds to the same region of a PDGF-B monomer(in the context of a PDGF-BB or PDGF-AB dimer) as an aptamer selectedfrom the aptamers shown in Table 1. In some embodiments, a PDGF aptamerbinds to the same region of a PDGF-B monomer (in the context of aPDGF-BB or PDGF-AB dimer) as an aptamer selected from the aptamers shownin Table 6. In some embodiments, a PDGF aptamer binds to the same regionof a PDGF-B monomer (in the context of a PDGF-BB or PDGF-AB dimer) asPDGF aptamer 4149-8_260. In some embodiments, a PDGF aptamer binds to aregion of PDGF-B comprising amino acids 24 to 86 of PDGF-B. In some suchembodiments, the PDGF aptamer competes for binding to PDGF with PDGFaptamer 4149-8_260. In some embodiments, a PDGF aptamer binds to PDGF-Bwith less than 15%, less than 14%, less than 13%, less than 12%, lessthan 11%, less than 10%, less than 9%, less than 8%, less than 7%, orless than 6% polar contacts to protein contact atoms. Polar contacts aredefined as the sum of hydrogen bonds and charge-charge interactions. Insome embodiments, a PDGF aptamer binds to PDGF-B with a ratio of polarcontacts to interface area of less than 0.01, less than 0.009, less than0.008, less than 0.007, or less than 0.006. In some embodiments, a PDGFaptamer binds to the same region of a PDGF-B monomer (in the context ofa PDGF-BB or PDGF-AB dimer) as PDGF aptamer 5169-4_26.

In some embodiments, a PDGF aptamer has any combination of the followingcharacteristics:

-   -   (a) binds to a region of PDGF-B comprising amino acids 24 to 86        of PDGF-B;    -   (b) competes for binding to PDGF with PDGF aptamer 4149-8_260;    -   (c) competes for binding to PDGF with PDGF aptamer 5169-4_26;    -   (d) binds to PDGF-B with a ratio of polar contacts to interface        area of less than 0.01, less than 0.009, less than 0.008, less        than 0.007, or less than 0.006; and/or    -   (e) binds to PDGF-B with less than 15%, less than 14%, less than        13%, less than 12%, less than 11%, less than 10%, less than 9%,        less than 8%, less than 7%, or less than 6% polar contacts to        protein contact atoms.

The PDGF aptamer can be selected to have any suitable dissociationconstant (K_(d)) for PDGF. In some embodiments, a PDGF aptamer has adissociation constant (K_(d)) for PDGF of less than 30 nM, less than 25nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 9 nM,less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, lessthan 4 nM, less than 3 nM, less than 2 nM, or less than 1 nM.Dissociation constants may be determined with a binding assay using amulti-point titration and fitting the equationy=(max−min)(Protein)/(K_(d)+Protein)+min as described in Example 3,below. In some embodiments, the PDGF aptamer is an aptamer with a K_(d)that is less than or equal to the K_(d) of an aptamer shown in any oneof Tables 1, 2 or 6 to 9. In some embodiments, the PDGF aptamer is anaptamer with a K_(d) that is less than or equal to the K_(d) of anaptamer shown in Table 1 or Table 6.

Aptamer 4149-8_1 binds in a 1:1 stoichiometry with a PDGF monomer. SincePDGF forms a tight homodimer that is required for reaction with itstarget receptors, a more efficient inhibition of PDGF activity might beachieved by using a dimeric or other multimeric form of aptamer4149-8_1. Thus, in some embodiments, the PDGF aptamer is amultimerization of any combination of the sequences of aptamer 4149-8_1,4149-8_379, and SEQ ID NOS 500 to 512. In some embodiments, an aptamerconstruct comprises a first aptamer selected from any of the PDGFaptamers described herein, and a second aptamer comprising any of thePDGF aptamers described herein, wherein the first aptamer and the secondaptamer may be the same or different. The first aptamer and the secondaptamer of the PDGF aptamer construct may be covalently or noncovalentlylinked. Nonlimiting exemplary linkages are known in the art and/or aredescribed herein. In some embodiments, a PDGF aptamer construct may becapable of binding two PDGF monomers simultaneously. In someembodiments, a PDGF aptamer construct binds PDGF with an affinity(K_(d)) of less than 10 nM.

Exemplary VEGF Aptamers

The VEGF aptamers of the instant disclosure were identified using theimproved SELEX method for identifying aptamers having slow off-rates asdescribed in Example 7, which describes a representative method for theselection and production of an aptamer that binds VEGF with a slowdissociation rate.

We truncated a clone from a Nap-dU VEGF-121 SELEX experiment to aminimal sequence of 29 nucleotides. This SOMAmer binds to both VEGF-121and VEGF-165 with high affinity (K_(d) values of 90 pM and 20 pM,respectively). The SOMAmer also potently inhibits the ability of bothVEGF isoforms to induce VEGFR2 phosphorylation in human umbilical veinendothelial cells in vitro (see Example 9), supporting the notion thatit binds to and blocks the receptor-binding domain on VEGF.

The present disclosure provides the first identification of aninhibitory aptamer to VEGF-121. Thus, the present VEGF aptamersrepresent broad inhibitors of VEGF, similar to protein-based drugs likebevacizumab (Avastin®), ranibizumab (Lucentis®) and aflibercept (Eylea®)(Papadopoulos et al. (2012) Angiogenesis 15:171; Yu et al. (2011)Biochem. Biophys. Res. Commun. 408:276. Thus, the present VEGF aptamersmay more effectively inhibit VEGF signaling than Macugen®, which is aselective inhibitor of VEGF-165.

A truncated clone from the successful Nap-dU VEGF-121 SELEX experimentprovided a sequence of 29 nucleotides. This aptamer (or SOMAmer)(4867-31 binds to both VEGF-121 and VEGF-165 with high affinity (K_(d)values of 90 pM and 20 pM, respectively). This SOMAmer also potentlyinhibits the ability of both VEGF isoforms to induce VEGFR2phosphorylation in human umbilical vein endothelial cells in vitro,supporting the concept that it binds to and blocks the receptor-bindingdomain on VEGF.

Aptamer 4867-31_192 binds in a 1:1 stoichiometry with a VEGF monomer.Since VEGF forms a tight homodimer that is required for reaction withits target receptors, a more efficient inhibition of VEGF activity mightbe achieved by using a dimeric or other multimeric form of aptamer4867-31_192. Thus, in some embodiments, the VEGF aptamer is amultimerization of any combination of the sequences of aptamer4867-31_192, SEQ ID NOS 513 to 516. In some embodiments, an aptamerconstruct comprises a first aptamer selected from any of the VEGFaptamers described herein, and a second aptamer comprising any of theVEGF aptamers described herein, wherein the first aptamer and the secondaptamer may be the same or different. The first aptamer and the secondaptamer of the VEGF aptamer construct may be covalently or noncovalentlylinked. Nonlimiting exemplary linkages are known in the art and/or aredescribed herein. In some embodiments, a VEGF aptamer construct may becapable of binding two VEGF monomers simultaneously. In someembodiments, a VEGF aptamer construct binds VEGF with an affinity(K_(d)) of less than 10 nM.

In some embodiments, a VEGF aptamer binds VEGF-121 with a K_(d) of lessthan 10 nM. In some embodiments, the VEGF aptamer comprises one or moremodified nucleotides. In some embodiments, the VEGF aptamer comprisesone or more modified nucleotides comprising hydrophobic modification. Insome embodiments, the VEGF aptamer comprises one or more modifiedpyrimidines. In some embodiments, the VEGF aptamer comprises one or moremodified pyrimidines shown in FIG. 12. In some embodiments, the VEGFaptamer comprises one or more modified pyrimidines shown in FIG. 12,groups II to V. In some embodiments, the VEGF aptamer comprises one ormore modified pyrimidines shown in FIG. 12, groups III to V. In someembodiments, the VEGF aptamer comprises one or more modified pyrimidinesshown in FIG. 12, groups III and IV. In some embodiments, the VEGFaptamer comprises one or more(N-naphthylmethylcarboxyamide)-2′-deoxyuridines (NapdUs).

In some embodiments, a VEGF aptamer comprises the sequence:

(SEQ ID NO. 513) 5′-GZZQAAEZECZZEZDRGAZZZAAAZGG-3wherein each Z is a modified pyrimidine;Q is selected from any modified or unmodified nucleotide and asubstituted or unsubstituted C₂-C₅₀ linker, or is absent;each E is independently selected from a G and a substituted orunsubstituted C₂-C₅₀ linker;D is selected from A and a substituted or unsubstituted C₂-C₅₀ linker,andR is selected from any modified or unmodified nucleotide and asubstituted or unsubstituted C₂-C₅₀ linker.

In some embodiments, a VEGF aptamer comprises a sequence selected from:

(SEQ ID NO. 514) 5′-CGZZQAAEZECZZEZDRGAZZZAAAZG-3′; (SEQ ID NO. 513)5′-GZZQAAEZECZZEZDRGAZZZAAAZGG-3′; (SEQ ID NO. 515)5′-CGZZQAAEZECZZEZDRGAZZZAAAZGG-3′; and (SEQ ID NO. 516)5′-CCGZZQAAEZECZZEZDRGAZZZAAAZGG-3′;wherein Z, Q, E, D, and R are as defined above.

In some embodiments, Z is a modified uridine. In some embodiments, eachZ is independently selected from the C-5 modified pyrimidines as definedherein. In some embodiments, each Z is independently selected from:

-   5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),-   5-(N-benzylcarboxyamide)-2′-O-methyluridine,-   5-(N-benzylcarboxyamide)-2′-fluorouridine,-   5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU),-   5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine (ThdU),-   5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),-   5-(N-tyrosylcarboxyamide)-2′-deoxyuridine (TyrdU),-   5-(N-3,4-methylenedioxybenzylcarboxyamide)-2′-deoxyuridine (MBndU),-   5-(N-4-fluorobenzylcarboxyamide)-2′-deoxyuridine (FBndU),-   5-(N-3-phenylpropylcarboxyamide)-2′-deoxyuridine (PPdU),-   5-(N-imidizolylethylcarboxyamide)-2′-deoxyuridine (ImdU),-   5-(N-isobutylcarboxyamide)-2′-O-methyluridine,-   5-(N-isobutylcarboxyamide)-2′-fluorouridine,-   5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),-   5-(N—R-threoninylcarboxyamide)-2′-deoxyuridine (TbrdU),-   5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,-   5-(N-tryptaminocarboxyamide)-2′-fluorouridine,-   5-(N-[1-(3-trimethylamomium) propyl]carboxyamide)-2′-deoxyuridine    chloride,-   5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU),-   5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,-   5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine,-   5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine),-   5-(N-2-naphthylmethylcarboxyamide)-2′-deoxyuridine (2NapdU),-   5-(N-2-naphthylmethylcarboxyamide)-2′-O-methyluridine,-   5-(N-2-naphthylmethylcarboxyamide)-2′-fluorouridine,-   5-(N-1-naphthylethylcarboxyamide)-2′-deoxyuridine (NEdU),-   5-(N-1-naphthylethylcarboxyamide)-2′-O-methyluridine,-   5-(N-1-naphthylethylcarboxyamide)-2′-fluorouridine,-   5-(N-2-naphthylethylcarboxyamide)-2′-deoxyuridine (2NEdU),-   5-(N-2-naphthylethylcarboxyamide)-2′-O-methyluridine,-   5-(N-2-naphthylethylcarboxyamide)-2′-fluorouridine,-   5-(N-3-benzofuranylethylcarboxyamide)-2′-deoxyuridine (BFdU),-   5-(N-3-benzofuranylethylcarboxyamide)-2′-O-methyluridine,-   5-(N-3-benzofuranylethylcarboxyamide)-2′-fluorouridine,-   5-(N-3-benzothiophenylethylcarboxyamide)-2′-deoxyuridine (BTdU),-   5-(N-3-benzothiophenylethylcarboxyamide)-2′-O-methyluridine, and-   5-(N-3-benzothiophenylethylcarboxyamide)-2′-fluorouridine.

In some embodiments, each Z is independently selected from the modifiedpyrimidines shown in FIG. 12, groups II to V. In some embodiments, eachZ is independently selected from the modified pyrimidines shown in FIG.12, groups III to V. In some embodiments, at least one, at least two, atleast three, at least four, at least five, at least six, at least seven,or at least eight Zs are5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU). In someembodiments, each Z is independently selected from the modifiedpyrimidines shown in FIG. 12, groups III to IV. In some embodiments,each Z is 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU).

A C₂-C₅₀ linker or spacer may be a backbone comprising a chain of 2 to50 carbon atoms (C₂-C₅₀) (saturated, unsaturated, straight chain,branched or cyclic), 0 to 10 aryl groups, 0 to 10 heteroaryl groups, and0 to 10 heterocyclic groups, optionally comprising an ether (—O—)linkage, (e.g., one or more alkylene glycol units, including but notlimited to one or more ethylene glycol units —O—(CH₂CH₂O)—; one or more1,3-propane diol units —O—(CH₂CH₂CH₂O)—, etc.); an amine (—NH—) linkage;an amide (—NC(O)—) linkage; and a thioether (—S—) linkage; etc.; whereineach backbone carbon atom may be independently unsubstituted (i.e.,comprising —H substituents) or may be substituted with one or moregroups selected from a C₁ to C₃ alkyl, —OH, —NH₂, —SH, —O—(C₁ to C₆alkyl), —S—(C₁ to C₆ alkyl), halogen, —OC(O)(C₁ to C₆ alkyl), —NH—(C₁ toC₆ alkyl), and the like. In some embodiments, a C₂-C₅₀ linker is aC₂-C₂₀ linker, a C₂-C₁₀ linker, a C₂-C₈ linker, a C₂-C₆ linker, a C₂-C₅linker, a C₂-C₄ linker, or a C₃ linker, wherein each carbon may beindependently substituted as described above.

In some embodiments, each substituted or unsubstituted C₂-C₅₀ linker isindependently selected from a substituted or unsubstituted C₂-C₂₀linker, a substituted or unsubstituted C₂-C₁₀ linker a substituted orunsubstituted C₂-C₈ linker, a substituted or unsubstituted C₂-C₆ linker,a substituted or unsubstituted C₂-C₅ linker, a substituted orunsubstituted C₂-C₄ linker, and a substituted or unsubstituted C₃linker. In some embodiments, each substituted or unsubstituted C₂-C₅₀linker is a substituted or unsubstituted C₂-C₁₀ linker. In some suchembodiments, each substituted or unsubstituted C₂-C₁₀ linker is asubstituted or unsubstituted C₂-C₅ linker, a substituted orunsubstituted C₂-C₆ linker, a substituted or unsubstituted C₂-C₅ linker,a substituted or unsubstituted C₂-C₄ linker, or a substituted orunsubstituted C₃ linker.

In some embodiments, one or more nucleosides of a VEGF aptamer comprisea modification selected from a 2′-position sugar modification (such as a2′-amino (2′-NH₂), a 2′-fluoro (2′-F), or a 2′-O-methyl (2′-OMe)), amodification at a cytosine exocyclic amine, an internucleoside linkagemodification, and a 5-methyl-cytosine. In some embodiments, a VEGFaptamer comprises a 3′ cap, a 5′ cap, and/or an inverted deoxythymidineat the 3′ terminus.

In some embodiments, a VEGF aptamer comprises at least one modifiedinternucleoside linkage. In some embodiments, at least one, at leasttwo, at least three, at least four, or at least five internucleosidelinkages are phosphorothioate linkages.

In some embodiments, a VEGF aptamer has a sequence selected from thesequences shown in Tables 10 to 14. In some embodiments, a VEGF aptamerhas a sequence selected from the sequences shown in Tables 10 to 14 thathave a K_(d) of less than 10 nM. In some embodiments, a VEGF aptamer hasa sequence that is at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% identical to the sequences shown in Tables 10to 14. In some embodiments, a VEGF aptamer has a sequence that is atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical to the sequences shown in Tables 10 to 14 that have aK_(d) of less than 10 nM. Percent identity is determined as describedabove for PDGF aptamers, except that the reference sequences are theVEGF aptamer sequences shown in Tables 10 to 14, such as the sequencesthat have a K_(d) of less than 10 nM.

In some embodiments, the present disclosure provides a VEGF aptamerthat, upon binding VEGF, modulates a VEGF function. In some embodiments,a VEGF aptamer inhibits VEGF-mediated phosphorylation of a VEGFreceptor, such as VEGFR1 or VEGFR2. In some embodiments, a VEGF aptamerinhibits VEGF-mediated phosphorylation of VEGF receptor. In variousembodiments, the VEGF aptamer modulates a VEGF function in vivo, such asinhibiting VEGF-mediated receptor phosphorylation in vivo. In variousembodiments, the VEGF aptamer has a sequence selected from the sequencesshown in Tables 10 to 14. In various embodiments, the VEGF aptamer isselected from the aptamers shown in Tables 10 to 14 that have a K_(d) ofless than 10 nM. In various embodiments, the VEGF aptamer is selectedfrom the aptamers shown in Tables 10 to 14. In some embodiments, theVEGF aptamer comprises at least 12, at least 13, at least 14, at least15, at least 16, at least 17, at least 18, at least 19, at least 20, atleast 21, at least 21, at least 22, at least 23, at least 24, at least25, at least 26, at least 27, at least 28, at least 29, or at least 30contiguous nucleotides of a sequence shown in Tables 10 to 14 that havea K_(d) of less than 10 nM. In some embodiments, a VEGF aptamer consistsof at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 21, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, atleast 27, at least 28, at least 29, or at least 30 contiguousnucleotides that are identical in nucleobase sequence to a sequenceshown in Tables 10 to 14 that have a K_(d) of less than 10 nM. In someembodiments, the VEGF aptamer comprises at least 12, at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least19, at least 20, at least 21, at least 21, at least 22, at least 23, atleast 24, at least 25, at least 26, at least 27, at least 28, at least29, or at least 30 contiguous nucleotides of an aptamer shown in Tables10 to 14. In some embodiments, the VEGF aptamer comprises at least 12,at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 21, at least 21, at least22, at least 23, at least 24, at least 25, at least 26, at least 27, atleast 28, at least 29, or at least 30 contiguous nucleotides of anaptamer shown in Tables 10 to 14 that have a K_(d) of less than 10 nM.In some embodiments, a VEGF aptamer consists of at least 12, at least13, at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 21, at least 22, at least23, at least 24, at least 25, at least 26, at least 27, at least 28, atleast 29, or at least 30 contiguous nucleotides of an aptamer shown inTables 10 to 14. In some embodiments, a VEGF aptamer consists of atleast 12, at least 13, at least 14, at least 15, at least 16, at least17, at least 18, at least 19, at least 20, at least 21, at least 21, atleast 22, at least 23, at least 24, at least 25, at least 26, at least27, at least 28, at least 29, or at least 30 contiguous nucleotides ofan aptamer shown in Tables 10 to 14 that have a K_(d) of less than 10nM.

In any of the embodiments herein, a VEGF aptamer may comprise additionalnucleotides or other chemical moieties on the 5′ end, the 3′ end, orboth the 5′ and the 3′ end of the aptamer.

The VEGF aptamer can contain any number of nucleotides in addition tothe VEGF binding region. In various embodiments, the VEGF aptamer caninclude up to about 100 nucleotides, up to about 95 nucleotides, up toabout 90 nucleotides, up to about 85 nucleotides, up to about 80nucleotides, up to about 75 nucleotides, up to about 70 nucleotides, upto about 65 nucleotides, up to about 60 nucleotides, up to about 55nucleotides, up to about 50 nucleotides, up to about 45 nucleotides, upto about 40 nucleotides, up to about 35 nucleotides, up to about 30nucleotides, up to about 25 nucleotides, or up to about 20 nucleotides.

In some embodiments, the VEGF aptamer is selected from an aptamer thathas similar binding characteristics and ability to treat VEGF associatedatherosclerosis, macular degeneration, fibrosis, and cancer conditionsas an aptamer shown in Tables 10 to 14 have a K_(d) of less than 10 nM.In some embodiments, a VEGF aptamer binds to the same region of VEGF-121as an aptamer selected from the aptamers shown in Tables 10 to 14 thathave a K_(d) of less than 10 nM. In some embodiments, a VEGF aptamerbinds to the same region of a VEGF-121 as a VEGF aptamer shown in Table10, 11, 12, 13 or 14 that have a K_(d) of less than 10 nM. In someembodiments, a VEGF aptamer binds to the same region of VEGF-121 as VEGFaptamer 4867-31_183.

The VEGF aptamer can be selected to have any suitable dissociationconstant (K_(d)) for VEGF. In some embodiments, a VEGF aptamer has adissociation constant (K_(d)) for VEGF-121 of less than 30 nM, less than25 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 9nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, lessthan 4 nM, less than 3 nM, less than 2 nM, or less than 1 nM.Dissociation constants may be determined with a binding assay using amulti-point titration and fitting the equationy=(max−min)(Protein)/(K_(d)+Protein)+min as described in Example 3,below.

In some embodiments, an aptamer construct comprises a first aptamerselected from any of the VEGF aptamers described herein, and a secondaptamer comprising any of the VEGF aptamers described herein, whereinthe first aptamer and the second aptamer may be the same or different.The first aptamer and the second aptamer of the VEGF aptamer constructmay be covalently or noncovalently linked. Nonlimiting exemplarylinkages are known in the art and/or are described herein. In someembodiments, a VEGF aptamer construct may be capable of binding two VEGFmonomers simultaneously. In some embodiments, a VEGF aptamer constructbinds VEGF with an affinity (K_(d)) of less than 10 nM.

Exemplary PDGF/VEGF Aptamer Constructs

There is considerable evidence that more efficient blocking oftumor-associated and ocular angiogenesis, coupled with new blood vesselregression, is possible with combined inhibition of VEGF and PDGF-Bsignaling pathways (Bergers, G., et al. (2003) J. Clin. Invest.111:1287; Jo, N., et al. (2006) Am. J. Pathol. 168:2036). This effect ismediated by the disruption of tight cell-cell association betweenendothelial cells, which form initial capillary sprouts, andperiendothelial cells (or pericytes), which encircle the new bloodvessels as they mature, rendering the blood vessels less susceptible toVEGF inhibitors (Benjamin, L. E., et al. (1998) Development 125:1591;Benjamin, L. E., et al. (1999) J. Clin. Invest. 103:159). The aptamersdescribed herein can form the basis of such a dual inhibitor.

In some embodiments, a PDGF/VEGF aptamer construct comprises any of thePDGF aptamers described herein linked to any of the VEGF aptamersdescribed herein. In some embodiments, a PDGF/VEGF aptamer constructcomprises any of the PDGF aptamers shown in Table 1 linked to any of theVEGF aptamers shown in Table 10 to 14 that have a K_(d) less than 10 nM.The linkage may be covalent or noncovalent.

The PDGF/VEGF aptamer construct may comprise a PDGF aptamer and a VEGFaptamer in any orientation, such as a PDGF aptamer linked at or near its3′ end to a point at or near the 5′ end of a VEGF aptamer, or a VEGFaptamer linked at or near its 3′ end to a point at or near the 5′ end ofa PDGF aptamer, or any other orientation that preserves the bindingproperties of each aptamer of the construct.

In some embodiments in which the linkage is covalent, the PDGF/VEGFaptamer construct may be linked through a phosphate or phosphorothioatelinkage. Many other covalent linkages are also contemplated, such aslinkages through various linker moieties, including, but not limited to,hexaethylene glycol linkers, polyethylene glycol linkers, substituted orunsubstituted hydrocarbon linkers, etc. One skilled in the art canselect a suitable covalent linkage for linking a PDGF aptamer to a VEGFaptamer.

In some embodiments, the PDGF aptamer and the VEGF aptamer are linkedvia a noncovalent linkage. Noncovalent linkages include, but are notlimited to, biotin/streptavidin; metal-binding peptides/metals;hybridizable modified and/or unmodified oligonucleotides; etc. One ofordinary skill in the art can select a suitable noncovalent linkage forlinking a PDGF aptamer to a VEGF aptamer.

Pharmaceutical Compositions Comprising Aptamers and Aptamer Constructs

In some embodiments, pharmaceutical compositions comprising at least oneaptamer or aptamer construct described herein and at least onepharmaceutically acceptable carrier are provided. Suitable carriers aredescribed in “Remington: The Science and Practice of Pharmacy,Twenty-first Edition,” published by Lippincott Williams & Wilkins, whichis incorporated herein by reference. Pharmaceutical compositions thatinclude at least one aptamer or aptamer construct described herein andat least one pharmaceutically acceptable carrier may also include one ormore active agents that is not a PDGF or VEGF inhibitor.

The aptamers described herein can be utilized in any pharmaceuticallyacceptable dosage form, including, but not limited to, injectable dosageforms, liquid dispersions, gels, aerosols, ointments, creams,lyophilized formulations, dry powders, tablets, capsules, controlledrelease formulations, fast melt formulations, delayed releaseformulations, extended release formulations, pulsatile releaseformulations, mixed immediate release and controlled releaseformulations, etc. Specifically, the aptamers described herein can beformulated: (a) for administration selected from any of oral, pulmonary,intravenous, intra-arterial, intrathecal, intra-articular, rectal,ophthalmic, colonic, parenteral, intracisternal, intravaginal,intraperitoneal, local, buccal, nasal, and topical administration; (b)into a dosage form selected from any of liquid dispersions, gels,aerosols, ointments, creams, tablets, sachets and capsules; (c) into adosage form selected from any of lyophilized formulations, dry powders,fast melt formulations, controlled release formulations, delayed releaseformulations, extended release formulations, pulsatile releaseformulations, and mixed immediate release and controlled releaseformulations; or (d) any combination thereof.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can comprise one or more of the followingcomponents: (1) a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; (2) antibacterial agents such as benzylalcohol or methyl parabens; (3) antioxidants such as ascorbic acid orsodium bisulfite; (4) chelating agents such asethylenediaminetetraacetic acid; (5) buffers such as acetates, citratesor phosphates; and (5) agents for the adjustment of tonicity such assodium chloride or dextrose. The pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. A parenteral preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic.

Pharmaceutical compositions suitable for injectable use may includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition should be sterile and should be fluid to theextent that easy syringability exists. The pharmaceutical compositionshould be stable under the conditions of manufacture and storage andshould be preserved against the contaminating action of microorganismssuch as bacteria and fungi. The term “stable”, as used herein, meansremaining in a state or condition that is suitable for administration toa subject.

The carrier can be a solvent or dispersion medium, including, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion, and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol or sorbitol, and inorganic saltssuch as sodium chloride, in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activereagent (e.g., an aptamer, and/or an aptamer construct) in anappropriate amount in an appropriate solvent with one or a combinationof ingredients enumerated above, as desired, followed by filteredsterilization. Generally, dispersions are prepared by incorporating atleast one aptamer, and/or aptamer construct into a sterile vehicle thatcontains a basic dispersion medium and any other desired ingredient. Inthe case of sterile powders for the preparation of sterile injectablesolutions, exemplary methods of preparation include vacuum drying andfreeze-drying, both of which will yield a powder of an aptamer, and/oran aptamer construct plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

In some embodiments, an aptamer, and/or an aptamer construct isformulated for intravitreal injection. Suitable formulations forintravitreal administration are described, e.g., in. Ocular drugdelivery is discussed, e.g., in Rawas-Qalaji et al. (2012) Curr. EyeRes. 37: 345; Bochot et al. (2012) J. Control Release 161:628; Yasukawaet al. (2011) Recent Pat. Drug Deliv. Formul. 5:1; and Doshi et al.(2011) Semin. Ophthalmol. 26:104. In some embodiments, a pharmaceuticalcomposition comprising an aptamer, and/or an aptamer construct isadministered by intravitreal injection once per week, once per twoweeks, once per three weeks, once per four weeks, once per five weeks,once per six weeks, once per seven weeks, once per eight weeks, once pernine weeks, once per 10 weeks, once per 11 weeks, once per 12 weeks, orless often than once per 12 weeks.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed, for example, in gelatin capsules orcompressed into tablets. For the purpose of oral therapeuticadministration, the aptamer, and/or aptamer construct can beincorporated with excipients and used in the form of tablets, troches,or capsules. Oral compositions can also be prepared using a fluidcarrier for use as a mouthwash, wherein the compound in the fluidcarrier is applied orally and swished and expectorated or swallowed.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from a pressured container or dispenser thatcontains a suitable propellant, e.g., a gas such as carbon dioxide, anebulized liquid, or a dry powder from a suitable device. Fortransmucosal or transdermal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the active reagents are formulated into ointments, salves, gels, orcreams, as generally known in the art. The reagents can also be preparedin the form of suppositories (e.g., with conventional suppository basessuch as cocoa butter and other glycerides) or retention enemas forrectal delivery.

In some embodiments, an aptamer, and/or an aptamer construct is preparedwith a carrier that will protect against rapid elimination from thebody. For example, a controlled release formulation can be used,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc.

Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

Additionally, suspensions of an aptamer, and/or an aptamer construct maybe prepared as appropriate oily injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils, such as sesame oil,or synthetic fatty acid esters, such as ethyl oleate, triglycerides, orliposomes. Non-lipid polycationic amino polymers may also be used fordelivery. Optionally, the suspension may also include suitablestabilizers or agents to increase the solubility of the compounds andallow for the preparation of highly concentrated solutions.

In some cases, it may be especially advantageous to formulate oral orparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of an aptamerand/or aptamer construct calculated to produce the desired therapeuticeffect in association with the required pharmaceutical carrier. Thespecification for the dosage unit forms of aptamers and/or constructsdescribed herein are dictated by and directly dependent on thecharacteristics of the particular aptamer and/or aptamer construct andthe particular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active agent for thetreatment of individuals.

Pharmaceutical compositions comprising at least one aptamer, and/oraptamer construct can include one or more pharmaceutical excipients.Examples of such excipients include, but are not limited to, bindingagents, filling agents, lubricating agents, suspending agents,sweeteners, flavoring agents, preservatives, buffers, wetting agents,disintegrants, effervescent agents, and other excipients. Suchexcipients are known in the art. Exemplary excipients include: (1)binding agents which include various celluloses and cross-linkedpolyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PH101and Avicel® PH102, silicified microcrystalline cellulose (ProSolvSMCC™), gum tragacanth and gelatin; (2) filling agents such as variousstarches, lactose, lactose monohydrate, and lactose anhydrous; (3)disintegrating agents such as alginic acid, Primogel, corn starch,lightly crosslinked polyvinyl pyrrolidone, potato starch, maize starch,and modified starches, croscarmellose sodium, cross-povidone, sodiumstarch glycolate, and mixtures thereof; (4) lubricants, including agentsthat act on the flowability of a powder to be compressed, and includingmagnesium stearate, colloidal silicon dioxide, such as Aerosil® 200,talc, stearic acid, calcium stearate, and silica gel; (5) glidants suchas colloidal silicon dioxide; (6) preservatives, such as potassiumsorbate, methylparaben, propylparaben, benzoic acid and its salts, otheresters of parahydroxybenzoic acid such as butylparaben, alcohols such asethyl or benzyl alcohol, phenolic compounds such as phenol, orquaternary compounds such as benzalkonium chloride; (7) diluents such aspharmaceutically acceptable inert fillers, such as microcrystallinecellulose, lactose, dibasic calcium phosphate, saccharides, and/ormixtures of any of the foregoing; examples of diluents includemicrocrystalline cellulose, such as Avicel® PH101 and Avicel® PH102;lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose®DCL21; dibasic calcium phosphate such as Emcompress®; mannitol; starch;sorbitol; sucrose; and glucose; (8) sweetening agents, including anynatural or artificial sweetener, such as sucrose, saccharin sucrose,xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame; (9)flavoring agents, such as peppermint, methyl salicylate, orangeflavoring, Magnasweet® (trademark of MAFCO), bubble gum flavor, fruitflavors, and the like; and (10) effervescent agents, includingeffervescent couples such as an organic acid and a carbonate orbicarbonate. Suitable organic acids include, for example, citric,tartaric, malic, fumaric, adipic, succinic, and alginic acids andanhydrides and acid salts. Suitable carbonates and bicarbonates include,for example, sodium carbonate, sodium bicarbonate, potassium carbonate,potassium bicarbonate, magnesium carbonate, sodium glycine carbonate,L-lysine carbonate, and arginine carbonate. Alternatively, only thesodium bicarbonate component of the effervescent couple may be present.

In various embodiments, the formulations described herein aresubstantially pure. As used herein, “substantially pure” means theactive ingredient (e.g., an aptamer, and/or an aptamer construct) is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition). In someembodiments, a substantially purified fraction is a composition whereinthe active ingredient comprises at least about 50 percent (on a molarbasis) of all macromolecular species present. Generally, a substantiallypure composition will include more than about 80% of all macromolecularspecies present in the composition. In various embodiments, asubstantially pure composition will include at least about 85%, at leastabout 90%, at least about 95%, or at least about 99% of allmacromolecular species present in the composition. In variousembodiments, the active ingredient is purified to homogeneity(contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of a single macromolecular species.

Kits Comprising Aptamers and Aptamer Constructs

The present disclosure provides kits comprising any of the aptamers,and/or aptamer constructs described herein. Such kits can comprise, forexample, (1) at least one aptamer, and/or aptamer constructs; and (2) atleast one pharmaceutically acceptable carrier, such as a solvent orsolution. Additional kit components can optionally include, for example:(1) any of the pharmaceutically acceptable excipients identified herein,such as stabilizers, buffers, etc., (2) at least one container, vial orsimilar apparatus for holding and/or mixing the kit components; and (3)delivery apparatus.

Methods of Treatment

The present disclosure provides methods of preventing or treating (e.g.,alleviating one or more symptoms of) medical conditions through the useof a PDGF aptamer or aptamer construct, a VEGF aptamer or aptamerconstruct, and/or a VEGF/PDGF aptamer construct. The methods compriseadministering a therapeutically effective amount of such aptamers and/oraptamer constructs to a subject in need thereof. The described aptamerscan also be used for prophylactic therapy. In some embodiments, theaptamer and/or aptamer construct is administered orally orintravenously.

The aptamer and/or aptamer construct used in methods of treatment canbe: a PDGF aptamer or aptamer construct a VEGF aptamer or aptamerconstruct, and/or a VEGF/PDGF aptamer construct described herein, or apharmaceutically acceptable salt thereof, or a prodrug thereof.

The individual or subject can be any animal (domestic, livestock orwild), including, but not limited to, cats, dogs, horses, pigs andcattle, and preferably humans. As used herein, the terms patient,individual, and subject may be used interchangeably.

As used herein, “treating” describes the management and care of apatient for the purpose of treating a disease, condition, or disorderand includes the administration of an aptamer, and/or an aptamerconstruct to prevent the onset of the symptoms or complications of adisease, condition or disorder, to alleviate symptoms or complicationsof the disease, condition, or disorder; or to eliminate the presence ofthe disease, condition or disorder in the patient. More specifically,“treating” includes reversing, attenuating, alleviating, minimizing,suppressing or halting at least one deleterious symptom or effect of adisease (disorder) state, disease progression, disease causative agentor other abnormal condition. Treatment is generally continued as long assymptoms and/or pathology ameliorate.

As used herein, “preventing” means preventing in whole or in part;ameliorating or controlling; reducing, lessening, or decreasing; orretarding or halting.

In various embodiments, the disclosed compositions and methods are usedto treat cardiovascular diseases, cancers, fibrosis, renal diseases orophthalmic diseases.

In some embodiments, the disclosed compounds or pharmaceuticallyacceptable salts thereof, or prodrugs, can be administered incombination with other treatments that improve or eradicate the diseaseconditions as described above. Compositions including the disclosedaptamers and/or aptamer constructs may contain, for example, more thanone aptamer. In some examples, a composition containing one or moreaptamers is administered in combination with another usefulcardiovascular agent or anticancer agent or antifibrotic agent etc. Ingeneral, the currently available dosage forms of the known therapeuticagents for use in such combinations will be suitable.

“Combination therapy” (or “co-therapy”) includes the administration ofan aptamer and/or aptamer construct composition and at least one secondagent as part of a specific treatment regimen intended to provide thebeneficial effect from the co-action of these therapeutic agents. Thebeneficial effect of the combination includes, but is not limited to,pharmacokinetic or pharmacodynamic co-action resulting from thecombination of therapeutic agents. Administration of these therapeuticagents in combination typically is carried out over a defined timeperiod (usually minutes, hours, days or weeks depending upon thecombination selected).

“Combination therapy” may, but generally is not, intended to encompassthe administration of two or more of these therapeutic agents as part ofseparate monotherapy regimens that incidentally and arbitrarily resultin the combinations of the present disclosure. “Combination therapy” isintended to embrace administration of these therapeutic agents in asequential manner, that is, wherein each therapeutic agent isadministered at a different time, as well as administration of thesetherapeutic agents, or at least two of the therapeutic agents, in asubstantially simultaneous manner. Substantially simultaneousadministration can be accomplished, for example, by administering to thesubject a single dose having a fixed ratio of each therapeutic agent orin multiple, single doses for each of the therapeutic agents.

The dosage regimen utilizing the aptamers and/or aptamer constructs isselected in accordance with a variety of factors, including, forexample, type, species, age, weight, gender and medical condition of thesubject; the severity of the condition to be treated; the route ofadministration; the renal and hepatic function of the subject; and theparticular aptamer and/or aptamer constructs or salts thereof employed.An ordinarily skilled physician or veterinarian can readily determineand prescribe the effective amount of the composition required toprevent, counter or arrest the progress of the condition.

In general, the dosage, i.e., the therapeutically effective amount,ranges from about 1 pig to about 100 mg/kg body weight of the subjectbeing treated, per day.

EXAMPLES

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention as defined by theappended claims. All examples described herein were carried out usingstandard techniques, which are well known and routine to those of skillin the art. Routine molecular biology techniques described in thefollowing examples can be carried out as described in standardlaboratory manuals, such as Sambrook et al. (2001) Molecular Cloning: ALaboratory Manual, 3rd. ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.).

Example 1. PDGF Aptamer Selection and Sequences

Preparation of Candidate Mixtures:

A candidate mixture of partially randomized ssDNA oligonucleotides wasprepared by polymerase extension of a DNA primer annealed to abiotinylated ssDNA template.

SELEX Conditions:

Aptamers to the PDGF-BB protein (R&D Systems) were selected by SomaLogicInc, as described (Gold et al. (2010) PLoS One 5:e15004), from a librarycontaining a 40-nucleotide random region in which Bn-dU was substitutedfor dT. The forward primer was 5′-CGCCCTCGTCCCATCTC, and the reverseprimer was 5′-CGTICTCGGTGGTGTIC. The PDGF-BB protein was biotinylatedand partitioned on streptavidin MyOne-SA (Dynal) beads. Preferentialselection of aptamers with slow dissociation rates was achieved using akinetic challenge wherein protein-DNA complexes were incubated in thepresence of 10 mM dextran sulfate at 37° C. with increased incubationtimes and decreased protein concentrations in successive rounds. Kineticchallenge was initiated in round 4 of the selection and was continuedthrough the final 8^(th) round with incubation times as follows: 5minutes round 4, 15 minutes rounds 5-7, 30 minutes round 8.

Pool Sequencing:

Oligonucleotide sequences from the 8^(th) round pool were cloned andseveral clones were sequenced. This led to the identification of afamily of related sequences, as exemplified by the 4149-8_1.

Deep Sequencing of PDGF SELEX Pool:

To evaluate more completely the sequences within the 4149-8_1 aptamerfamily, the 8^(th) round pool was sequenced using 454 pyrosequencingtechnology. The pool DNA was amplified with 454 primers and the PCRproduct was purified and normalized using a Sequal normalization plate(Invitrogen, Cat # A10510-01). The eluate was run on a gel to confirmthe size and purity of each amplicon. The purified PCR product wassequenced at the 454 pyrosequencing facility at the University ofColorado Health Science Center in Aurora Colo.

The 454 sequences were aligned with 4149-8_1 by CLUSTAL analysis. Thesequence data set from the pool contained 10,803 full length sequences(i.e., those sequences containing both primer sequences) of which 3,839were unique. These 3,839 unique sequences were searched for the motif“5′-ZACNCGCGZZZAZAGCG” (identity=0.65) and then “ZZ” (identity=1.0)upstream from this. There were 436 sequences found that contained bothof these motifs. In addition, 58 other sequences contained only thefirst pattern but with a generally low identity and with no evidenthairpin structure upstream. The 436 sequences were then aligned asfollows, (1) with respect to “ZZ”, (2) with respect to the center of theloop, and (3) with respect to “ZACNCGCGZZZAZAGCG”. For all thesequences, the percentage identity at each position with 4149-8_1 wascalculated as listed in FIG. 3A. Tables 1 and 2 lists a number ofsequences representative of the 4149-8_1 aptamer family of sequences.

Aptamer Synthesis:

The modified deoxyuridine-5-carboxamide amidite reagents used forsolid-phase synthesis were prepared by: condensation of5′-O-(4,4′-dimethoxytrityl)-5-trifluoroethoxycarbonyl-2′-deoxyuridine(Nomura et al. (1997) Nucl. Acids Res. 25:2784) with the appropriateprimary amine (RNH₂,1.2 eq; Et₃N, 3 eq.; acetonitrile; 60° C.; 4 h);3′-O-phophitidylation with2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (1.2 eq.; iPr₂EtN, 3eq.; CH₂Cl₂; −10 to 0° C.; 4 h); and purification by flashchromatography on neutral silica gel (Still, et al. (1978) J. Org. Chem.43:2923). Aptamers were prepared by solid phase synthesis using thephosphoramidite method (Beaucage and Caruthers (1981) Tetrahedron Lett.22:1859) with some adjustments to the protocol to account for the uniquebase modifications described herein. Detritylation was accomplished with10% dichloroacetic acid in toluene for 45 seconds; coupling was achievedwith 0.1 M phosphoramidites in 1:1 acetonitrile:dichloromethaneactivated by 5-benzylmercaptotetrazole and allowed to react 3 times for5 minutes; capping and oxidation were performed according to instrumentvendor recommendations. Deprotection was effected with 1:1:2,t-butylamine:methanol:water (Mullah 1998), reacted for 24 hours at 37degrees centigrade. Aptamers were synthesized at 200 nmol scale andpurified from a polyacrylamide gel using UV shadowing as described(Fitzwater and Polisky (1996) Methods Enzymol. 267:275) with CostarSpin-X (not including siliconized glass wool or spun polypropyleneprefilter) and Amicon YM3 concentration per manufacturer'srecommendations.

Modified Nucleotide Structure Activity Relationship and AffinityMaturation:

To examine the contribution of each of the eight benzyl side chains tobinding, we performed another series of systematic point substitutionsby chemically synthesizing 5-position variants with a custom-madelibrary of modified dU phosphoramidites. For this purpose, we designed alibrary to allow us to probe the microenvironment of each of thepositions by varying the size, polarity, disposition of H-bond donorsand acceptors, linker length, and orientation of the 5-positionsubstituents. In choosing the functional groups for this analysis, weaimed to include variations on a theme of the original modification (inthis case, the benzyl group), amino acid side chains overrepresented incomplementarity determining regions (CDRs) of antibodies (liketryptophan and tyrosine) (Mian, I S, et al. (1991) J. Mol. Biol.217:133; Ramaraj T. et al. (2012) Biochim. Biophys. Acta. 1824:520), and“privileged” fragments of small-molecule drugs (Welsch et al. (2010)Curr. Opin. Chem. Biol. 14:347). In a sense, we endeavored to combineelements of affinity maturation in antibodies and structure-activityrelationship (SAR) optimization in medicinal chemistry. Although weutilized a single modified nucleotide during SELEX, post-SELEXoptimization is constrained only by the synthetic accessibility of themodified monomers and compatibility with solid-phase synthesis.

The effect of individual substitutions of the benzyl group with fourteenalternative moieties at the 5-position is summarized in FIGS. 1C and D,and FIG. 6B, with relative affinities expressed as dissociation constantratios and relative PDGF Rβ phosphorylation expressed as percentphospho-PDGF Rβ ratios. Substitution with dT, which only has a methylgroup at the 5-position, represents the most drastic change, and in thatsense is comparable to alanine scanning mutagenesis in proteins(Cunningham, B. C. et al. (1989) Science 243:1330). Not surprisingly,this was the least tolerated substitution at six of the eight modifiednucleotide positions. The exceptions were nucleotides 1 and 7, wherethis substitution was well-tolerated. These two positions also toleratedmany other substitutions, with some replacements yielding up to 5-foldimprovement in binding affinity (FIG. 6B). In contrast, nucleotides 8,17 and 18 exhibited the highest sensitivity to changes. The best singlesubstitutions were then combined, yielding additional variants including4149-8_255 and 4149-8_260 (FIG. 6B). Aptamer 4149-8_260, which combinedphenethyl-dU (Pe-dU) at nucleotide 17 and thiophene-dU (Th-dU) atnucleotide 18, showed excellent binding to both PDGF-BB and PDGF-AB(FIG. 6B). It is worth noting that the affinity of the originallyselected SOMAmer was already so high (K=20 pM) that it approached thedetection limit of the binding assay, so it is possible that the degreeof affinity improvement is underestimated. We have applied similarpost-SELEX optimization strategies to other SOMAmers with weaker initialbinding (e.g., K_(d) values ranging from 100 pM to >10 nM), and haveobserved affinity improvements of up to 100-fold.

Homodimers of PDGF Aptamer 4149-8_260 (SL5):

Since PDGF forms a covalently linked homodimer, and two SOMAmers bind toeach PDGF homodimer, we determined the effect on binding ofhomodimerized SOMAmers. The affinity of the PDGF aptamer homodimerscould be substantially improved compared to the affinity of thecorresponding monomers, due to avidity effects. The crystal structureshowed that the 5′ ends of the SOMAmer were 38 Å apart, while the 3′ends were 74 Å apart. Connecting the 5′ to 3′ end would require at least63 Å since the shortest path between the two points bisected theprotein. Two types of homodimers were ordered, based on readilyavailable chemistry. These were 1) head-to-tail homodimers connected bytwo to six Heg linkers, which provide ˜20 Å distance per Heg, and 2)3′-3′ homodimers connected via a synthetic doubler support, combinedwith one to three Hegs. The homodimers of 4149-8_260 were tested in thePDGF-BB Zorbax binding assay. The binding assay was performed withlimiting amount of SOMAmer, and would not distinguish binding of oneSOMAmer per protein dimer versus binding of two SOMAmers per proteindimer. The structure of the homodimers is shown in Table 1 (sequences4149-8_334 through 4149-8_342). The K_(d) values obtained in the Zorbaxassay suggested that in the 5′ to 3′ configuration, a longer linker wasdesirable, and gave up to 10-fold improvement in binding affinity, asshown in Table 1a. In the 3′-3′ linked homodimers, the shorter linkeractually appeared to perform better than the longer linker. This wascorroborated by cellular phosphorylation results, see Table 1a.

Based on these sequences, an exemplary consensus sequence is:

(SEQ ID NO: 502) 5′-ZZVCL_(n)GV′ZACNMGCGZZZAZAGCG-3′,whereinV is selected from an A, C or G;V′ is selected from a C, G or Z, wherein V′ is complementary to V;N is independently selected from any naturally occurring or modifiednucleotide;M is selected from a C or A;Z is independently selected from a modified pyrimidine; L is a spacerselected from any naturally occurring or modified nucleotide, ahydrocarbon linker, a polyethylene glycol linker or a combinationthereof; andn is 0 to 20;wherein one or more nucleotide insertions are optionally included.

Sequence Truncation Studies:

Systematic truncation from the 5′ and 3′ ends of 4149-8_1 was performedto define a minimum length required to retain full binding activity ofthe aptamer to human PDGF-BB, as shown in Table 3. K_(d) values for asubset of the truncations are shown. Z=Benzyl-deoxyuridine (Bn-dU); A,C, G and T are deoxyribonucleotides.

Protein Expression and Purification, and Aptamer Complex Formation

For crystallography studies, recombinant human PDGF-BB protein waspurchased from Creative BioMart (Shirley, N.Y.). The recombinant proteinwas expressed in E. coli cells. Aptamer solutions were thawed and thenannealed by heating to ˜95° C. for 5 minutes, then incubating at 40° C.for 5 minutes, then cooling to room temperature. The annealed aptamersolution was mixed with protein at a 1.1:1 ratio of DNA to protein. Thecomplex was diluted 5-fold in buffer containing 20 mM Na/K phosphate (pH7) and 100 mM NaCl. The resulting mixture was concentrated to ˜4 mg/mLin protein in a 1.5 mL Amicon centrifugal filter. Final concentrationwas estimated from the final volume of retentate.

Example 2. Crystallization and Structure of PDGF-Aptamer Complex

Crystals were grown using the sitting drop vapor diffusion method inCompact, Jr. plates (Emerald BioSystems, WA) set up at 16° C. Crystalsfor data collection were obtained from a primary screen (ProPlex,Molecular Dimensions). The crystal for the PDGF-BB:4149-8_255 complexwas grown from 100 mM magnesium acetate, 100 mM sodium acetate (pH 4.5)and 8% (w/v) PEG 8000. The crystal for the PDGF-BB:4149-8_260 complexwas grown from 100 mM magnesium acetate, 100 mM sodium cacodylate (pH6.5) and 15% (w/v) PEG 6000. Crystals were harvested with Litho Loopsand cryoprotected by quick transfer to reservoir solution containing 33%(v/v) ethylene glycol before flash-cooling by plunging directly intoliquid nitrogen.

Data Collection and Structure Determination:

Data for both structures were collected at beamline 19-ID of theAdvanced Photon Source (Argonne, Ill.). The data sets were processedusing XDS (Kabsch 2010). The structure of the PDGF-BB:4149-8_260 complexwas initially phased by molecular replacement using Phaser from the CCP4software suite (CCP4, 1994) with the protein model of PDGF from thestructure of the PDGF-BB:Beta-type PDGF receptor complex (PDB entry3MJG) as the search model. Molecular replacement located a singleprotein monomer per asymmetric unit. Inspection of the electron densitymaps following an initial round of restrained refinement in REFMACshowed features consistent with nucleic acid adjacent to the proteinmodel. The model of the aptamer was subsequently built through a processof “bootstrapping”, i.e., partial models were subjected to iterativerounds of refinement; resulting in marginally improved maps whichallowed further model building. First, phosphate ions were built innucleic acid backbone density. Secondly, phosphates were replaced by dTresidues. Following refinement, modified residues could be discerned byprotrusions of positive difference electron density. Identification ofthe modified residues facilitated determination of the sequence registerof the aptamer, and in the final steps dT residues were replaced withthe correct nucleobases. All manual building was performed using theCrystallographic Object-Oriented Toolkit (Coot) (Emsley & Cowtan, 2004).The structure of the PDGF-BB:4149-8_255 structure was solved bymolecular replacement using the finished model of the 4149-8_260complex.

In each structure, a protuberance of electron density was observedcontiguous with the electron density of the Oγ atom of residues Thr88and Thr90. Although O-mannosylation has been reported at these sites forrecombinant PDGF-B expressed in yeast (Settineri, et al., (1990)) thereis little reason to expect similar post-translational modifications inthe PDGF-B expressed in E. coli. As the observed electron densitysuggested less than full occupancy, the threonine residues were modeledwithout any post-translational modification.

Table 4 discloses data-collection statistics and refinement and modelstatistics of two aptamer ligands with 4149-8_260 (SEQ ID. NO. 211) and4149-8_255 (SEQ ID. NO. 207) respectively.

Table 5 illustrates base-pair parameters for the PDGF BB aptamercompared to B-form DNA. The PDGF BB aptamer adopts deviated B-formconformations in the 5′ stem loop domain and in both stems of theminiknot. Where appropriate, mean values and standard deviations (inparenthesis) are given. Aptamer values are based on analysis usingweb3DNA (Zheng et al. (2009) Nucleic. Acids Res. 37:W240) and B-DNAvalues (as found in high resolution crystal structures) were determinedusing 3DNA as described and reported in Olson, et al. (2001) J. Mol.Biol. 313(1): 229.

The monomeric subunits of PDGF-BB form twisted f-sheets that dimerize inan anti-parallel orientation characteristic of the cystine knot familyof proteins (Oefner et al. (1992) EMBO J. 11:3921). SL5 (4149-8_260)binds two homologous sites at either end of the long axis, crossing thehomodimer interface and contacting each of the three PDGF loops (FIG.7A). The SOMAmer is composed of two domains connected by a network ofhydrophobic aromatic interactions (FIG. 7B). At the 5′ end, a short stemis capped with a Heg loop (disordered in the crystal structure), whilethe remainder of the molecule folds into an extraordinarily small H-typepseudoknot (Aalberts, D. P. et al. (2005) Nucleic Acids Res. 33:2210),with modified nucleotides clustering at the stem loop/pseudoknotjunction. Remarkably, all eight modified nucleotides are in contact withPDGF. Seven modified nucleotides cluster together along a hydrophobicgroove on the protein, while Bn-dU adopts an extended conformation,following a channel at the PDGF homodimer interface. Two naturalnucleotides also contact PDGF, with the remaining natural nucleotidescontributing to internal structure (FIG. 2 and FIG. 7). The secondarystructure elements of SL5, a stem-loop and a pseudoknot, are well-knownnucleic acid structural motifs. However, replacement of certainconventional bases with modified nucleotides offers novel functionalgroups for alternative interactions. This distinguishing feature of SL5results in an extensive hydrophobic surface for protein binding as wellas unique intramolecular contacts between canonical and modifiednucleotides.

Although the 3′ end of SL5 exhibits hallmark characteristics of anH-type pseudoknot (Staple, D. W. et al. (2005) PLoS Biol. 3:e213), thiscategorization understates the unconventional nature of this signature“miniknot” motif. Compared to the smallest structurally reported H-typepseudoknot which requires 21 nucleotides (Nonin-Lecomte S. et al. (2006)Nucleic Acids Res. 34:1847), the SL5 miniknot consists of a mere 16nucleotides (FIG. 7B). Furthermore, deletion of the terminal mG24:dC12base pair of stem 2 (S2) results in undiminished binding affinity (FIG.3), demonstrating the functional integrity of a 14-nucleotide miniknot.With unprecedented backbone twists and stacking interactions, theminiknot represents a novel pseudoknot variant in which unusually smallsize is attained via stabilization contributed by packing of thehydrophobic moieties of the modified nucleotides.

Miniknot stem 1 (S1) formally consists of just two Watson-Crick basepairs (FIG. 8A), while loop 2 (L2) is composed nominally of 5 bases,Pe-dU17, Th-dU18, dA19, Bn-dU20 and mA21. While interactions between L2and S1 are a defining feature of pseudoknots, they are typically limitedto H-bonding. In contrast, the SOMAmer miniknot makes atypicalloop-to-stem stacking interactions, supported by unconventional basepairing. In particular, S1 is stabilized by stacking with anon-canonical Bn-dU17:Bn-dU20 base pair derived from L2 (FIG. 8C andFIG. 8B), effectively creating a three base-pair S1 with a novelbackbone discontinuity. In contrast to previously described U:U iminocarbonyl base pairs, the Pe-dU17:Bn-dU20 base pair utilizes a singleH-bond between N3 of Bn-dU17 and the carbonyl oxygen in the amide linkerof Bn-dU20 (FIG. 8D). The syn conformation about the glycosyl bond ofBn-dU20 impedes H-bonding with Bn-dU17, but allows Bn20 to stack withBn-dU8 base without steric clashing with the sugar of Bn-dU8. Theunconventional Pe-dU17:Bn-dU20 base pair is made possible by a 2800 turnin the backbone between nucleotides 18 and 20 (FIG. 8C). This dramaticstrand reversal allows Bn-dU20 base to stack with the sugar of dA9 andform a hydrogen bond with Pe-dU17. Importantly, the Pe-dU17:Bn-dU20 basepair derives additional stabilization through hydrophobic interactionsconferred by the modified nucleotides; the ethylene (linker) portion ofthe Pe-dU17 side chain is directed toward Bn16 (CH::π) while its benzylgroup is stacked in π-π edge-to-face interactions with Bn2 and Th18(FIG. 8E). One additional interaction between L2 and S1 is a base triple(mA21:dG15:dC10; FIG. 8F), a recurrent motif in pseudoknots (Chen, G. etal. (2009) Proc. Natl. Acad. Sci. USA 106:12706). This is the onlylong-range tertiary interaction in SL5 that does not involve themodified nucleotides.

Loop 1 (L1) consists of a single extruded nucleotide, mA11 that allowsthe backbone to make a tight 94° turn, with the intrastrand phosphatedistance between mA11 and dC12 compressing to just 5.9 Å (FIG. 8G).H-type pseudoknots often have one or two nucleotides in L, whichtypically form hydrogen bonds with S2 and stack into the helicaljunction (Nonin-Lecomte, S. et al. (2006) Nucleic Acids Res. 34:1847;Michiels, P. J. et al. (2001) J. Mol. Biol. 310:1109). The extruded L1nucleotide is necessary to keep the structure condensed so that the 5′stem domain can interface with the miniknot through the hydrophobicmoieties of the modified nucleotides. As expected, the extruded base isnot conserved (FIG. 3) and can be replaced with a single C3 spacer (FIG.6A), however, its deletion abrogates binding, presumably due tointerference with the miniknot formation.

The Watson-Crick base pairs of miniknot S1 (dA9:Bn-dU16, dC10:dG15)assemble by the favored H-type pseudoknot arrangement in which strandone of S2 leads directly into strand two of S1, providing efficientstacking of the stems (Klein, D. J. et al. (2009) Nat. Struct. Mol.Biol. 16:343). The three base pairs of S2 are composed entirely ofWatson-Crick interactions and form a slightly undertwisted B-form helix(FIG. 8H). This understwisting results in helical parameters that moreclosely resemble A-form helices, as expected for pseudoknot topology;however, the relevance of these calculations is equivocal, given theshort length of the helices in this structure. S2 does not form aconventional coaxial stack with S1 due to severe helical overwinding atthe junction (twist angle of 70°) formed by dC10:dG15 of S1 anddC14:dG22 of S2. Continuous stacking of the stems is neverthelessmaintained as dC14 stacks with dG15, and dG22 stacks with mA21 from thebase triple (FIG. 8H). The extensive helical twist at this junction isnecessary to allow mA21 to bridge the major groove of S2 whilebroadening the minor groove for base triple formation. Thisconfiguration is typical in pseudoknots with one or two nucleotides inL1 (Nonin-Lecomte, S. et al. (2006) Nucleic Acids Res. 34:1847;Michiels, P. J. et al. (2001) J. Mol. Biol. 310:1109).

The SL5 5′ stem is composed of two Watson-Crick base pairs (mA3:Bn-dU7and dC4:dG6) and a non-canonical Bn-dU2:Bn-dU8 base pair at the base ofthe stem (FIG. 8I). The Bn-dU2:Bn-dU8 base pair contains two hydrogenbonds, a typical 4-carbonyl-N3 and a unique 4-carbonyl from Bn-dU2 baseto amide linker of Bn-dU8 bond (FIG. 8J). Analysis of related sequencesin the affinity-enriched pool shows that the length and base compositionof the 5′ stem can change, with the notable exception of the invariantBn-dU:Bn-dU brace at the base of the stem (FIGS. 3A and B), highlightingthe importance of this non-canonical base pair in the overall structureand function of SL5. Stability of the 5′ stem helix is further bolsteredby stacking of dU8, Bn20 and Pro82 of PDGF (FIG. 9H). The 5′ stem-loopand miniknot domains of SL5 converge where the backbone makes a sharp111° bend. Significant twist angles and radial displacement of the basepairs in the 5′ stem results in bases 2-4 and 6-7 having greaterstacking overlap (because of helix undertwisting) than in conventionalB-form helices, while Bn-dU8 base is shifted out and Bn-dU7 base stackswith the amide linker of Bn-dU8 (FIG. 8I). This atypical helixfacilitates critical interactions with the rest of SL5 and with PDGF;Bn-dU8 base stacks with Bn20 while Bn8 lies perpendicularly between therings of Bn16 and Bn20 in consecutive π-π edge-to-face interactions.These long-range tertiary interaction define a precise hinge between theminiknot and the stem-loop domains (FIGS. 8L and 8K). The lack ofcurvature between the first two nucleotides prevents clashing of Bn-dUbase with Bn2, augmenting stacking of the rings (FIG. 8I). Bn2 sits inthe middle of a hydrophobic cluster created by Bn7 and Bn8 (from the 5′stem) and Bn16, Pel7 and Bn20 (from the miniknot) (FIG. 7B, FIG. 8I andFIG. 8K). This hydrophobic cluster contributes to stabilization of theSOMAmer, supported by the observation that SL5 exhibits a T_(m) of 64°C., which is >30° C. higher than its analog that lacks the modifiednucleotides.

In addition to SL5, we also solved the structure of SL4 (4149-8_255),which is identical to SL5 except for the replacement of Bn-dU8 withisobutyl-dU (iB-dU). When iB-dU8 was combined with Pe-dU17 and Th-dU18in variant SL4, the SOMAmer showed substantially weaker binding(˜20-50-fold vs. SL5) and a 75-fold lower in vitro inhibitory activity(FIG. 1A, FIG. 1B and FIG. 6B). The smaller non-aromatic isobutyl sidechain cannot form the energetically favorable π-π edge-to-face stackingseen with the benzyl side chains of Bn-dU20, Bn-dU8 and Bn-dU16 in SL5(FIG. 8M and FIG. 8N). This creates a hole in the center of thehydrophobic cluster at the protein interface, effectively unlocking thehinge between the 5′ stem and the miniknot domains. The structuraleffect of this substitution is directly analogous to a Phe to Leumutation in the hydrophobic core of a protein. Such protein mutationsare well-described (Kadonosono, T. et al. (2003) Biochemistry 42:10651;Lin, H. J. et al. (2003) Biochim. Biophys. Acta. 1649:16; Baase, W. A.et al. (2010) Protein Sci. 19:631) and usually have a significantdestabilizing effect. Junctions between secondary structure motifs areknown to play a critical role in determining nucleic acid tertiarystructure (Pyle, A. M. et al. (2011) Curr. Opin. Struct. Biol. 21:293).

Despite markedly weaker target-binding affinity, SL4 exhibits similarthermal melting profile to SL5 in the absence of ligand (T_(m) values of62° C. and 64° C., respectively). This is consistent with the notionthat the cavity and altered junction topology created by iB-dU8substitution in SL4 destabilizes the protein-binding interface, whileleaving the intradomain structures of the SOMAmer intact. Theconformations of free SOMAmers in solution may well be very differentfrom those in the complex with the protein, which could also diminishthe relationship between T_(m) and binding affinity. In fact, since theenergetic cost of solvating a large hydrophobic surface of the SOMAmeris likely to be substantial, we expect the uncomplexed SOMAmer tocollapse around the hydrophobic side chains and adopt a conformation inwhich the hydrophobic side chains are partially protected from thesolvent.

In contrast to previously described protein:aptamer complexes,hydrophobic interactions dominate the interface between SL5 and PDGF(FIG. 2, FIG. 4 and FIG. 9). Binding to PDGF-BB creates a buried surfacearea of ˜1225 Å² per SOMAmer. The eight modified nucleotides of SL5create an extensive hydrophobic interface that interacts with 13non-polar amino acids of PDGF (Ala35, Phe37, Leu38, Val39, Trp40, Pro42,Cys52, Cys53, Ile75, Ile77, Pro82, Ile83, and Phe84), which account forapproximately half of the total non-polar contacts, with the remaindercomprising aliphatic regions of polar or charged amino acids such asGlu24, Arg27, Asn36, Asn54, Asn55, Arg56, Arg73, Lys74, Lys80, Lys85,and Lys86 (FIG. 9). Similar interactions between completely non-polarresidues and non-polar moeities of charged amino acids are oftenobserved in proteins. Thus, the structural diversity afforded by themodified nucleotides in SOMAmers enables them to mimic the richrepertoire of interactions accessible to proteins. The strikingdifference in the extent of hydrophobic contacts made by the SOMAmercompared to traditional aptamers is evident when the interface atoms aredisplayed on the surfaces of the target proteins. SL5 exhibitsremarkably few polar interactions, having just six H-bonds and onecharge-charge interaction with PDGF (FIG. 4), despite close proximity tobasic amino acids. Relative to the contact surface area, this issignificantly lower than what is typical for aptamers. The total numberof H-bonds and charge-charge interactions (that is, polar contacts) forsix traditional aptamers increases approximately linearly in directproportion to the interface area (FIG. 10A, FIG. 22A) with a correlationcoefficient of 0.91 and an average of 1.9-0.4 polar contacts per 100 Å²interface area. SL5, as well as two additional SOMAmers in otherco-crystal structures, clearly fall outside of the 99% confidenceintervals of this trend, with less than half the number of polarcontacts per interface area (average of 0.7-0.2 per 100 Å² interfacearea), while exhibiting a trend toward higher binding affinities fortheir targets (FIG. 22C). In terms of ligand efficiency (free energy ofbinding per non-hydrogen contact atom) (Kuntz, I. D. et al. (1999) Proc.Natl. Acad. Sci. USA 96:9997), aptamers and SOMAmers do not appear to bedifferent (FIG. 22C), encompassing a range of values observed withprotein-based and small molecule-based ligands (Wells, J. A. et al.(2007) Nature 450:1001). Free energies of binding per interface area arealso similar (FIG. 22C). What is different, however, is the value offree energy of binding per polar contact, which is about twice as largefor SOMAmers than for aptamers (FIGS. 22 B and C), consistent with thenotion that SOMAmers derive a larger contribution to binding fromhydrophobic interactions.

Charge-charge interactions often contribute less than 0.2 kcal/mol tothe stability of a folded protein (Sali, D. et al. (1991) J. Mol. Biol.220:779. In contrast, burying just a single methylene group is estimatedto contribute ˜1-1.5 kcal/mol to globular protein stability and/orbinding interactions (Kellis, J. T., Jr. et al. (1988) Nature 333:784;Pace, C. N. et al. (2011) Mol. Biol. 408:514). SOMAmer structures reveala strong reliance on hydrophobic interactions and in this sense, theirbinding to proteins more closely resemble typical protein-proteininteractions. Consistent with this observation, the affinity of SL5 forPDGF shows virtually no decrease across a broad range of saltconcentrations (0.1 to 1.0 M NaCl) or pH values (5.0 to 8.8), incontrast to the effects seen with traditional aptamers (Ahmad, K. M. etal. (2011) PLoS ONE 6:e27051; Tang, Q. et al. (2007) J. Colloid.Interface. Sci. 315:99).

Post-SELEX optimization facilitates fine tuning of shape complementaryand hydrophobic packing interactions. For example, the exceptional shapecomplementary of Pe-dU17 and Th-dU18 at the protein interface (FIG. 10B)is corroborated with the structure-activity relationships (FIG. 6B).Bn-dU1 also forms a unique interaction with PDGF-BB, with the benzylring sitting in a tunnel formed by the Cys43-Cys52 disulfide bond and asalt bridge between Glu24 of PDGF chain1 and Arg56 of chain 2 (FIG.10C). The crystal structure suggests that the binding pocket canaccommodate a variety of side chains, including larger bicyclicsubstituents, thereby enhancing this point of contact with the protein.Indeed, we have identified several modified nucleotide substitutions atthis position that confer 5- to 10-fold enhancement in binding affinity(FIG. 6B).

A notable feature of the PDGF-BB:SL5 structure is the degree to whichthe SOMAmer mimics PDGFRβ. The receptor binds to PDGF primarily throughhydrophobic interactions, including seven hydrophobic amino acids at thePDGF interface (Shim, A. H. et al. (2010) Proc. Natl. Acad. Sci. USA107:11307). SL5 binding site largely overlaps that of the receptor withthe Bn-dU aromatic rings occupying the same hydrophobic groove on theprotein (FIG. 11). PDGF contacts both the receptor and SL5 with 24residues, of which 10 are shared. These shared or “promiscuous” residueslikely represent a hot spot of binding energy on the surface of PDGF(Wells, J. A. et al. (2007) Nature 450:1001; Clackson, T. et al. (1994)Science 267:383. However, compared with PDGF Rβ, SL5 exhibits a 10-foldhigher affinity for PDGF-BB (Lokker, N. A. et al. (1997) J. Biol. Chem.272: 33037). Consistent with these observations, SL5 is a potentinhibitor of PDGF-BB (FIG. 1B and FIG. 6B).

Example 3. Binding Affinity Assays

For determination of target binding affinity, SOMAmers were 5′end-labeled using T4 polynucleotide kinase (New England Biolabs) andγ-³²P-ATP (Perkin Elmer). Binding assays were performed by incubatingradiolabeled SOMAmer (˜20,000 c.p.m) at a concentration of ˜0.03-0.05 nMand target protein at concentrations ranging from 10⁻⁷ to 10⁻¹² M in1×SB18T buffer (40 mM HEPES, pH 7.5; 120 mM NaCl; 5 mM KCl; 5 mM MgCl₂and 0.01% TWEEN-20) at 37° C. for 30 minutes. Bound complexes were mixedwith Zorbax resin and captured on Durapore filter plates. The fractionof SOMAmer bound was quantified with a PhosphorImager (FUJI FLA-3000).Raw binding data were corrected for nonspecific background binding ofradiolabeled SOMAmer to Zorbax resin. Equilibrium dissociation constants(K_(d)) was determined as previously described (Jellinek et al. (1993)Proc. Natl. Acad. Sci. 91:11227). Competitor tRNA at a concentration of200 nM was included in isoform specificity studies as indicated in FIG.5. To determine the salt dependence on the PDGF-BB/SOMAmer and E10030interactions, binding affinity assays were performed and analyzed asdescribed above in the presence of 40 mM Hepes pH 7.5, 0.01% TWEEN-20and either 100 mM, 250 mM, 500 mM, 750 mM or 1.0 M NaCl). The log-logplots of the salt concentrations versus the dissociation constants werefit using simple linear regression. The slope of the plots representsthe number of counter-ions released from the DNA upon protein binding,as described by the counter-ion condensation theory of (Manning, G. S.(1969) J. Chem. Phys. 51:924). The affinity of aptamer 4149-8_260 (SEQID NO. 211) for PDGF showed little change across a broad range of saltconcentrations (0.1 to 1.0 M NaCl) or pH values (5.0 to 8.8), incontrast to the effects seen with traditional aptamers (Ahmad, K. M. etal. (2011) PLoS One 6:e27051; Tang, Q. et al. (2007) J. Colloid.Interface Sci. 315: 99).

Example 4. PDGF-BB Cellular Phosphorylation Assay

PDGF-BB Activity.

For testing the ability of PDGF-BB SOMAmers to inhibit PDGF Rβactivation, Hs27 human foreskin fibroblast cells (American Type CultureCollection) were seeded at 5000 cells/well into a 96-well plate, andserum-starved for 24 hours. SOMAmers (varying concentrations asindicated in the figures) were incubated with PDGF-BB (20 ng/mL)(Creative BioMart) in serum-free media for 30 minutes at 37° C., thenthe complex was added to serum-starved Hs27 cells. At five minutes poststimulation, the supernatant was discarded and the cells were lysed inLysis Buffer #9 (R&D Systems: 1% NP-40 Alternative, 20 mM Tris (pH 8.0),137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM activated sodiumorthovanadate, 10 μg/mL Aprotinin, and 10 μg/mL Leupeptin) on ice for 5minutes. Elisa detection of phospho-PDGF Rβ was performed using theDuoSet Phospho-PDGF Rβ kit (R&D Systems) according to the manufacturer'sinstructions. The percent phospho-PDGF Rβ was measured at OD₄₅₀,corrected for plate absorbance and background signal with a no stimulantcontrol. Experiments were generally performed in duplicate ortriplicate. Data were plotted in GraphPad Prism 3.0 and fit to a onesite competition curve using nonlinear regression. Representative plotof the IC₅₀ determination is shown in FIG. 13 for SOMAmers 4149-8_379and 5′ amino-linker modified SOMAmer 4149-8_379, with IC₅₀ values of 1.6nM and 1.7 nM, respectively.

For activity screening of variants of clone 4149-8, percent inhibitionof PDGF-BB-induced PDGF Rβ phosphorylation in Hs27 fibroblasts, underthe same conditions as described above but at a single concentration ofSOMAmer variants (generally 20 nM), was evaluated.

Example 5. Additional PDGF Ligands Based on Nap-dU Modification

To identify additional aptamers that bind to PDGF-BB with high affinity,we have performed another SELEX experiment with a library comprisingNap-dU modified nucleotides. The selections were performed in a mannersubstantially analogous to the one described in Example 1 above andresulted in the identification of the Nap-dU aptamer clone 5169-4.

Deep Sequencing of PDGF Nap-dU SELEX Pool:

To evaluate more completely the sequences within the 5169-4_1 aptamerfamily, the 7^(th) round pool was sequenced using 454 pyrosequencingtechnology. The pool DNA was amplified with 454 primers and the PCRproduct was purified and normalized using a Sequal normalization plate(Invitrogen, Cat # A10510-01). The eluate was run on a gel to confirmthe size and purity of each amplicon. The purified PCR product wassequenced at the 454 pyrosequencing facility at the University ofColorado Health Sciences Center in Aurora, Colo.

The sequence data set from the pool contained 8,273 full lengthsequences (i.e., those sequences containing both primer sequences) ofwhich 1,629 were unique. These 1,629 unique sequences were used to findstatistically significant n-mer patterns by counting all possible n-mersin the sequence set, from 4-mers to 30-mers. By comparing the counts foreach identified n-mer to counts expected at random for n-mers from apool of the same composition, statistically significant patterns werefound. Two major patterns were identified in the sequence set and5169-4_1 was found aligned within the second pattern defined by theconserved sequence motif “APGPAPGCACAPCP” found in 11 sequences. Asearch through all unique sequences for this motif (identity=0.75) found51 sequences that were then aligned by the motif. For all the sequences,the fractional identity at each position in the alignment was calculatedas listed in FIG. 14, with the consensus sequence indicated.

Sequence Truncation Studies:

Systematic truncation from the 5′ and 3′ ends of the 50-nucleotide5169-4 clone was performed to define a minimum length required to retainfull binding activity of the aptamer to human PDGF-BB, as shown in Table6. K_(d) values for these truncations are shown (P=Napthyl-deoxyuridine(Nap-dU); A, C, G and T are deoxyribonucleotides). The 5169-4 cloneproved highly amenable to truncation and a 21-nucleotide sequence wasidentified (5169-4_26) that bound to PDGF-BB with improved bindingaffinity compared to the 50-mer (17 pM and 29 pM, respectively). The5169-4_26 21-mer contained 5 Nap-dU modified nucleotides versus 9 Nap-dUmodified nucleotides in the 50-mer.

C3 Spacer Single Substitutions in 5169-4_26 (21-mer):

The first round of post-SELEX modifications of the Nap-dU PDGF-BBaptamer included a C3 spacer walk at all positions in the 21-mer5169-4_26. The C3 spacer walk is meant to identify bases not requiredfor high affinity binding that could potentially be removed altogether,replaced with the C3 spacer or other linkers such as hexaethylene glycol(Heg) or polyethylene glycol (PEG) linkers. The results for the C3spacer substitutions are shown in Table 7. In this table, “P” denotesNap-dU, “C3” denotes C3 spacer, A, C and G denote deoxyribonucleotides,and “NB” denotes no binding up to 100 nM PDGF-BB. Three sites toleratedC3 substitution with a modest decrease in binding affinity: C1, G6 andC7 (numbering refers to the 21-mer, as shown below). One position, C15,tolerated a C3 spacer substitution with no effect on the bindingaffinity, compared to 5169-4_26.

2′-O-methyl Single Substitutions in 5169-4_26 (21-mer):

2′O-methyl substitutions were made at all natural bases in order toidentify positions that could tolerate this nuclease-resistantsubstitution. With the 2′O-methyl Nap phosphoramidite synthesized in ourlaboratories (Nap-mU), we also evaluated the Nap-dU positions that wouldtolerate Nap-mU single substitutions. In addition, deoxythymidine (T)was substituted for Nap-dU to assess the importance of each Nap-dU. Thebinding affinity results are shown in Table 8, and demonstrate that allpositions tolerated 2′O-methyl substitutions to varying degrees. Theeffect of 2′O-methyl substitutions at each deoxycytidine position (C)resulted in no change in the binding affinity, compared to 5169-4_26, upto a 2-fold decrease in binding affinity. The four deoxyguanosinepositions (G) showed varying results when substituted with 2′O-methylfrom 2.5-fold increased binding affinity at G14, to 5.5-fold decreasedbinding affinity at G10, compared to 5169-4_26. The 2′O-methylsubstitutions at the six deoxyadenosine positions (A) had from zero togreater than 50-fold adverse effect on the binding affinity. Thedeoxyadenosines towards the 5′ end of the aptamer (A3, A5 and A8) werethe three most sensitive to 2′O-methyl substitution. Only the Nap-dU atposition 21 completely tolerated Nap-mU substitution with no effect onthe binding affinity while the Nap-mU substitution at position 11 showeda 2.5-fold decrease in binding affinity, compared to 5169-4_26. Theremaining Nap-mU substitutions resulted in a 15- to 30-fold decrease inbinding affinity. The only substitutions that completed eliminatedbinding (at PDGF-BB concentrations up to 100 nM) were the deoxythymidinesubstitutions at positions 12 and 21, with the remaining deoxythymidinesubstitutions having a significant negative affect (>400 fold) on thebinding affinity. In Table 8, P=5-naphthalene modified dU, a superscript1 indicates a 2′-O-methyl modified nucleoside. A, C, G, and T representthe naturally occurring deoxyribonucleotides and “NB” denotes no bindingup to 100 nM PDGF-BB.

Multiple 2′-O-methyl Substitutions in 5169-4_26 (21-mer).

The combined effects of 2′-O-methyl substitutions in 5169-4_26 lead tothe identification of several variants with improved binding affinity,including variant 5169-4_146. This 21-mer has 11 positions that arenuclease-protected by 2′O-methyl and its binding affinity is at least20-fold greater than the parent truncate, 5169-4_26 (0.60 pM vs 17 pM,respectively). Many other variants also had significant improvements(approximately 3-fold) in binding affinity with combinations of 3 to 102′O-methyls. In Table 9, P=5-naphthalene modified dU, a superscript 1indicates a 2′-O-methyl modified nucleoside, A, C and G represent thenaturally occurring deoxyribonucleotides and “NB” denotes no binding upto 3.2 nM PDGF-BB.

Example 6. PDGF Nap-dU Aptamer Activity Assay

To analyze the inhibitory impact of PDGF Nap-dU aptamers on PDGF Rβactivation cellular phosphorylation inhibition assays were performed asdescribed in Example 4. The four aptamer sequences tested inhibited PDGFRβ activation with IC₅₀ values as follows: 5169-4_26, IC₅₀=1.6 nM;5169-4_84, IC₅₀=3.3 nM; 5169-4_85, IC₅₀=7.3 nM; 5169-4_112, IC₅₀=1.0 nM.

Example 7. VEGF Aptamer Selection and Sequences

Preparation of Candidate Mixtures: A candidate mixture of partiallyrandomized ssDNA oligonucleotides was prepared by polymerase extensionof a DNA primer annealed to a biotinylated ssDNA template.

VEGF SELEX Conditions:

Aptamers to recombinant human VEGF-121 protein (both from R&D Systems)were selected by SomaLogic Inc, as described (Gold et al. (2010) PloSOne 5:e15004), from a library containing a 40-nucleotide random regionin which Nap-dU was substituted for dT. For VEGF-121, the forward primerwas 5′-GCCACACCCTGCCCTC-3′ and the reverse primer was5′-GAGGACACAGACAGACAC-3′. VEGF-121 protein was biotinylated andpartitioned on streptavidin MyOne-SA (Dynal) beads. Preferentialselection of aptamers with slow dissociation rates was achieved using akinetic challenge wherein protein-DNA complexes were incubated in thepresence of 10 mM dextran sulfate at 37° C. with increased incubationtimes and decreased protein concentrations in successive rounds. In theVEGF-121 SELEX, rounds 4 and 5 included a 15 minute kinetic challengewhile rounds 6 and 7 (final round) included a 30 minute kineticchallenge.

The smallest alternatively spliced form of vascular endothelial growthfactor, VEGF-121, is a difficult protein target for SELEX. Withnaturally occurring DNA or RNA libraries, or with nucleic acid librariesmodified at the 2′-position of ribose, we have previously failed toobtain even a modest degree of affinity improvement. This is notable fortwo reasons. First, among members of the cystine knot superfamily,VEGF-121 has the highest structural similarity to PDGF-BB, with aroot-mean-square deviation of 1.9 Å for 124 Ca atoms (Muller et al.,1997). Second, the larger and the most prevalent VEGF isoform, VEGF-165,has proved to be a good target for SELEX. For example, pegaptanib(Macugen), the only aptamer-based therapeutic to have receivedregulatory approval to date (for the treatment of macular degeneration),binds only to VEGF-165 through the heparin-binding exon-7-encodeddomain, which is lacking in VEGF-121 (Lee et al., 2005; Ruckman et al.,1998). One difference between VEGF-121, VEGF-165 and PDGF-BB is theoverall charge, with pI values of 5.8, 8.5 and 10.1, respectively. Thispoints to the importance of polar interactions in aptamer binding.Successful affinity enrichment for VEGF-121 was ultimately achieved witha SELEX Nap-dU library.

Identification of VEGF-121 Nap-dU Aptamer Sequences:

Two highly related high affinity variants that differ at a singleposition (4867-15 and 4867-31) were identified from a Nap-dU SELEXexperiment performed as described above. Clone 4867-31 has beentruncated to a 29-mer in a series of deletion experiments (Table 7). Itis worth noting that truncation of both high affinity clones (4867-15and 4867-31) results in the same 29-mer since the single nucleotidedifference is outside the 5′ boundary of the minimal sequence. Truncatedvariants that encompass the shorter sequence with high affinity biding,29-mer 4867-31_143 (5′-CCGPP CAAGP GCPPG PAGGA PPPAA APGG-3′; where “P”is the single-letter designation for Nap-dU) and its close variants,bind to human VEGF-121, human VEGF-165, mouse VEGF-120 and rat VEGF-164with comparable affinities, ranging from 0.1-1 nM (Table 10). In thistable, “P” denotes Nap-dU; A, C, and G denote the naturally occurringdeoxyribonucleotides and “NB” denotes no binding up to 100 nM VEGF.

C3 Spacer Single Substitutions in 4867-15_2 (50-mer).

The first round of post-SELEX modifications of the VEGF-121 aptamer wasa C3 spacer walk at all positions in the 50mer 4867-15_2 (truncated50-mer). The C3 spacer walk is meant to identify bases not required forhigh affinity binding that could potentially be removed altogether,replaced with the C3 spacer or other linkers such as hexaethylene glycol(Heg) or polyethylene glycol (PEG) linkers. The results for the C3spacer substitutions are shown in Table 11. In this table, “P” denotesNap-dU, “V” denotes C3 spacer; A, C, and G denote the naturallyoccurring deoxyribonucleotides and “NB” denotes no binding up to 100 nMVEGF. At least three internal sites tolerated C3 substitution: C17, G26and G29 (numbering refers to the 50-mer, as shown below).

2′-O-methyl Single Substitutions in 4867-31_43 (32-mer).

2′O-methyl substitutions were made at natural bases in order to identifypositions that could tolerate this nuclease-resistant substitution. Inaddition, 2′-OMe-uridine (2′-OMeU) was substituted for Nap-dU to assessthe importance of each Nap-dU. In addition, C3 spacers were tested atcertain internal positions hypothesized to be extruded bases, now in thecontext of the 32mer. Internal deletions and alternative bases weretested at each of the three positions as well. The binding affinity andcell culture inhibition data for select SOMAmers (single concentrationof 20 nM) are shown below. The results are shown in Table 12, anddemonstrate that C8 (C17 in Table 11) did not completely toleratesubstitution to C3 in the context of the shorter truncate. The other twoputative extruded bases (G17 and G20) retain good binding and functionalactivity as C3 or alternative base substitutions in this context.Internal deletions at those positions were not tolerated. None of theNap-dU modifications could be replaced with 2′OMe-U in this experiment.Several internal sites tolerated 2′OMe modifications, however. In Table12, P=5-naphthalene modified dU, and a superscript 1 indicates a 2′-OMemodified nucleoside, V=3 carbon spacer, and an empty box indicates anucleoside deletion. A, C, G, and U represent the naturally occurringdeoxyribonucleotides.

2′-O-methyl Nap-dU Substitutions and Multiple 2′-O-methyl Substitutionsin 4867-31_143 (29-mer).

With the 2′O-methyl Nap phosphoramidite synthesized in our laboratories(Nap-mU), we evaluated the Nap-dU positions that would tolerate Nap-mUsingle substitutions. In addition, we tested combinations of 2′OMe andC3 linker substitutions at each of the natural bases. The bindingaffinity and cell culture inhibition data for select SOMAmers (singleconcentration of 20 nM) are shown below. As shown in Table 13 below,most of the Nap-dU residues did not tolerate OMe substitution, butsubstitution of Nap-mU for Nap-dU at position 22 gave a 10-fold increasein affinity, and excellent inhibitory activity. In this table,superscript “1” denotes 2′-O-methyl substitution, “V” denotes C3 spacer.The 2′-OMe combinations were mostly well-tolerated based on affinity,but certain combinations showed a striking loss of inhibitory activity.

The combined effects of 2′-O-methyl and C3 substitutions led to theidentification of several variants with improved binding affinity,including variant 4867-31_188. This 29-mer has 10 positions that arenuclease-protected by either 2′OMe or C3 and its binding affinity isabout 3-fold tighter than the parent truncate, 4867-31_143 (38 pM vs 140pM, respectively). Variant 4867-31_188 retains comparable cellularinhibition activity relative to the parent SOMAmer. See Table 13.

The only position that tolerated Nap-mU was nucleotide 22 (using4867-31_143 truncate as the parent sequence). This Nap-mU substitutionwas next placed into the background of the best combined 2′OMe SOMAmer,4867-31_188. In addition, the substitution of the original dG atposition 15 with a C3 spacer was compared with 2′-OMe substitution atthat position to examine the possibility that nuclease-protected basemight add rigidity to the molecule and hence increase binding. The bestaggregate result was obtained with variant 4867-31_192, which now has 9protected positions compared to the parent 29-mer truncated variant4867-31_143 (see Table 14 below; superscript “1” denotes 2′-O-methylsubstitution and “V” denotes C3 spacer).

Modified Nucleotide Structure Activity Relationship and AffinityMaturation:

To examine the contribution of each of the ten napthyl side chains tobinding, we performed another series of systematic point substitutionsby chemically synthesizing 5-position variants with a custom-madelibrary of modified dU phosphoramidites. For this purpose, we designed alibrary to allow us to probe the microenvironment of each of thepositions by varying the size, polarity, disposition of H-bond donorsand acceptors, linker length, and orientation of the 5-positionsubstituents. In choosing the functional groups for this analysis, weaimed to include variations on a theme of the original modification (inthis case, the napthyl group), amino acid side chains overrepresented incomplementarity determining regions (CDRs) of antibodies (liketryptophan and tyrosine) (Mian, I. S. et al. (1991) J. Mol. Biol.217:133; Ramaraj, T. et al. (2012) Biochim. Biophys. Acta. 1824:520),and “privileged” fragments of small-molecule drugs (17). FIG. 15 showsthe results of these substitutions, represented as the ratio of K_(d)values (substituted/unsubstituted). Of the 17 different modificationsubstitutions tested each of the ten Nap-dU positions, only foursubstitutions (Trp-dU 27, NE-dU 16, MBn-dU 10 and BT-dU 16) had littleto no effect of the binding affinity. All other substitutions resultedin weaker binding affinity, to varying degrees.

Deep Sequencing of VEGF SELEX Pool:

To evaluate more completely the sequences within the 4149-8_1 aptamerfamily, the enriched pool was sequenced using 454 pyrosequencingtechnology. The pool DNA was amplified with 454 primers and the PCRproduct was purified and normalized using a Sequal normalization plate(Invitrogen, Cat # A10510-01). The eluate was run on a gel to confirmthe size and purity of each amplicon. The purified PCR product wassequenced at the 454 pyrosequencing facility at the University ofColorado Health Sciences Center in Aurora, Colo.

The 454 sequences were aligned with 4867-31 by CLUSTAL analysis. Thesequence data set from the pool contained 13,139 full-length sequences(i.e., those sequences containing both primer sequences) of which 2,235were unique. These 2,235 unique sequences were searched for the motif5′-CCGPP CAAGP GCPPG PAGGA PPPAA APGG-3′. There were 86 sequences foundthat contained this motifs. For all the sequences, the percentageidentity at each position with 4867-31 was calculated as listed in FIG.16.

Example 8. VEGF Binding Affinity Assays

For determination of target binding affinity, SOMAmers were 5′end-labeled using T4 polynucleotide kinase (New England Biolabs) andγ-³²P-ATP (Perkin Elmer). Binding assays were performed by incubatingradiolabeled SOMAmer (˜20,000 c.p.m) at a concentration of ˜0.03-0.05 nMand target protein at concentrations ranging from 10⁻⁷ to 10⁻¹² M in1×SB18T buffer (40 mM HEPES, pH 7.5; 120 mM NaCl; 5 mM KCl; 5 mM MgCl₂and 0.01% TWEEN-20) at 37° C. for 30 minutes. Bound complexes were mixedwith Zorbax resin and captured on Durapore filter plates. The fractionof SOMAmer bound was quantified with a PhosphorImager (FUJI FLA-3000).Raw binding data were corrected for nonspecific background binding ofradiolabeled SOMAmer to Zorbax resin. Equilibrium dissociation constants(K_(d)) was determined as previously described (Jellinek et al. (1993)Proc. Natl. Acad. Sci. 90:11227).

Example 9. VEGF Activity Assay

To analyze the inhibitory impact of VEGF121 SOMAmers on the cellularkinase activity of VEGF-R2 (Vascular Endothelial Growth factor Receptor2), we used human umbilical vein endothelial cells (HUVECs) (Lonza,^(#)CC-2519) which expresses endogenously high level of VEGF-R2. HUVECcells were plated in EGM-2 (Endothelial Cell Growth Medium) supplementedwith EGM-2 BulletKit (^(#)CC-3162) containing 2% FBS, growth factors(hEGF, Hydrocortisone, VEGF, HFGF-B, R3-IGF-1), heparin, ascorbic acidand GA-1000 (Gentamicin, Amphotericin-B). When HUVEC cells reached 70 to80% confluence, they were plated in 24-well plate (10⁵ cells/well) andstarved overnight with serum-free medium.

SOMAmers (a single concentration at 20 nM or a range of concentrations)were added to the cultures with 20 ng/mL (1 nM) of VEGF-121 (R&D System,^(#)4464-VS) containing 1% BSA at 37° C. for 30 minutes. The cells werewashed in PBS two times and stimulated with the pre-incubatedVEGF-121/SOMAmers complex for 5 minutes. The treated cells were washedagain with PBS two times and added ice-cold lysis buffer (1% NP-40Alternative, 20 mM Tris (pH8.0), 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1mM activated sodium orthovanadate, 10 μg/mL Aprotinin and 10 μg/mLleupeptin supplemented with a Halt phosphatase inhibitor (ThermoScientific, ^(#)78428). The cell lysates were measured forphosphorylation of VEGF-R2 by using Human Phospho-VEGF R2/KDR Kit (R&D,DYC 1766-2).

In functional activity experiments in vitro, various truncated variantsof clone 4867-31 at a screening concentration of 20 nM are capable ofessentially completely inhibiting VEGFR2 phosphorylation induced byVEGF-121 or VEGF-165 (1-4 nM) in immortalized or primary human umbilicalvein endothelial cells (HUVECs). A representative plot of the IC₅₀determination is shown in FIG. 17 for VEGF aptamers 4867-31_43 and4867-31_192, with IC₅₀ values of 2.2 nM and 2.1 nM, respectively.

For activity screening of variants of clone 4867-31, we have evaluatedpercent inhibition of VEGF-induced VEGF R2 phosphorylation in HUVECs,under the same conditions as described above but at a singleconcentration of SOMAmer variants (generally 20 nM).

Example 10. Homodimer Constructs of PDGF and VEGF Aptamers

Both PDGF-BB and VEGF are disulfide linked homodimers that exert theirbiological effects by dimerizing their tyrosine kinase receptors leadingto receptor autophosphorylation and signal transduction. If more thanone aptamer can bind to its protein target, as is the case with PDGF-BBaptamer 4149-8_260 (based on the crystal structures), such aptamers canbe covalently linked in a multimeric construct in a manner that permitssimultaneous binding of individual aptamer subunits to the protein. Thiscan lead to improvement in affinity through avidity effect. Two types ofhomodimers were synthesized, based on readily available chemistry. Thesewere 1) head-to-tail homodimers connected by zero to six Heg linkers,which provide ˜20 Å distance per Heg, and 2) 3′-3′ homodimers connectedvia a synthetic doubler support, combined with one to three Hegs on eachside (that is, two, four or six Hegs total in the dimer). The homodimersof 4149-8_379, 5169-4_26 and 4867-31_192 were tested in a competitionbinding assay. For determination of competitor binding affinities,aptamer ligands were 5′ end-labeled using T4 polynucleotide kinase (NewEngland Biolabs) and γ-³²P-ATP (Perkin Elmer). Competition assays wereperformed by pre-mixing a fixed concentration of radiolabeled ligand(1.0 nM) with varying concentrations of competitor aptamer (10⁻¹¹ to10⁻⁶ M). The ligand and competitor dilutions were incubated with thetarget protein (100 pM) in 1×SB18T buffer (40 mM HEPES, pH 7.5; 120 mMNaCl; 5 mM KCl; 5 mM MgCl₂ and 0.01% TWEEN-20) at 37° C. for 60 minutes.Bound complexes were mixed with Zorbax resin and captured on Duraporefilter plates. The fraction of ligand bound was quantified with aPhosphorlmager (FUJI FLA-3000). Raw binding data were normalized tobinding without addition of competitor. Data were plotted in GraphPadPrism 3.0 and fit to a one site competition curve using nonlinearregression to determine the equilibrium dissociation constants for thecompetitor aptamers (K_(i)).

PDGF Homodimers:

The structure of the PDGF homodimers of sequences 4149-8_379 (sequences4149-8_438 through 4149-8_447) and 5169-4_26 (sequences 5169-4_134through 5169-4_143) are shown in Table 15. For the 4149-8_379 basedhomodimers, the K_(i) values obtained in the competition assay suggestedthat in the 5′ to 3′ configuration, a longer linker was desirable, sincea greater than 10-fold improvement in binding affinity was measured withfive Heg linkers compared to no Heg linker (0.25 pM vs 4.2 pM,respectively). In the 3′ to 3′ linked 4149-8_379 homodimers, the longerfour and six Heg linkers also performed at least 10-fold better than nolinker and approximately 2-fold better than the two Heg linkers. For the5169-4_26 based homodimers, the K_(i) values indicated a longer Heglinker was advantageous in the 5′ to 3′ configuration, since the K_(i)improved from 28 pM for no Heg linker to 3.6 pM for six Heg linkers.There was no difference in the K_(i) values for five and six Heg linkersin the 5′ to 3′ configuration. In the 3′ to 3′ linked 5169-4_26 basedhomodimers the same pattern was observed, with the K_(i) improving asthe Heg linker length increased. The six Heg linker showed a 5-foldimprovement in the K_(i) compared to no Heg linker (2.0 pM vs. 11 pM,respectively). In Tables 15 and 16, Z=Benzyl-deoxyuridine (Bn-dU),P=5-naphthalene modified dU (Nap-dU), M=methylenedioxybenzyl-dU(MBn-dU), a superscript 1 indicates a 2′-O-methyl modified nucleoside,no superscript indicates deoxyribonucleotides, “C3” indicates a threecarbon linker and “H” indicates a hexaethylene glycol linker.

Example 11. PDGF/VEGF Heterodimer Aptamer Constructs

Heterodimers Based on PDGF Aptamer 4149-8 and VEGF Aptamer 4867-31.

With the aim of developing constructs with specificity for PDGF andVEGF, we designed and tested a variety of aptamer constructs comprisinga VEGF aptamer linked to a PDGF aptamer. The first aptamer constructstested combined PDGF variant 4149-8_273 and VEGF 4867-31_183. Aptamerconstructs were synthesized head-to-tail, connected by zero to threehexaethylene glycol (Heg) linkers, in both orientations (either with thePDGF aptamer at the 5′ end or VEGF aptamer at the 5′ end). The resultsare shown in Table 17 and 18 below. In Table 17, “Z” denotes Bn-dU, “P”denotes Nap-dU, superscript “1” denotes 2′-O-methyl substitution, nosuperscript indicates deoxyribonucleotides, “V” denotes C3 spacer and“H” denotes hexaethylene glycol (Heg) linker. In Table 18, percentactivity remaining denotes fractional PDGF BR phosphorylation levels inHs27 fibroblasts in the presence of 20 nM aptamer relative to control(no aptamer).

Based on binding affinity for PDGF-BB, -AB, VEGF-121, and VEGF-165,aptamer construct 4149-8_320 appeared to give the best results in thisexperiment. We also tested the aptamer constructs in the PDGF cellularphosphorylation assay, as shown in Table 18. Based on the functionalassay data, all aptamer constructs tested inhibited PDGF-BB-induced PDGFBR phosphorylation in Hs27 fibroblasts. Aptamer constructs 4149-8_313,4149-8_314, 4149-8_315, 4149-8_316, 4149-8_319 and 4149-8_320 inhibitedPDGF-BB-induced PDGF βR phosphorylation with IC₅₀ values of <20 nM.Aptamer constructs 4149-8_317 and 4149-8_318 had IC₅₀ values of ˜20 nM.

We synthesized 4149-8_401, based on the configuration of 4149-8_320(5′PDGF-3Heg-VEGF3′), comprising PDGF aptamer 4149-8_379 and VEGFaptamer 4867-31_192. See Table 19. In this table, “Z” denotes Bn-dU, “P”denotes Nap-dU, M denotes MBn-dU, superscript “1” denotes 2′-O-methylsubstitution, no superscript denotes deoxyribonucleotides, “C3” denotesC3 spacer and “H” denotes hexaethylene glycol (Heg) linker. Aptamerconstruct 4149-8_401 showed binding affinity for PDGF-BB and VEGF121that was equivalent or better than the binding affinity of its precursoraptamer construct, 4149-8_320. See Table 20.

Aptamer constructs 4149-8_320 and 4149-8_401 inhibited PDGF-BB-inducedwere PDGF-Rβ phosphorylation in Hs27 fibroblasts with IC₅₀ values ofabout 1 nM and 5 nM, respectively. Further, aptamer construct 4149-8_401comprising a 5′ amino linker conjugated to either 20 kDa or 40 kDa PEGmaintained the ability to inhibit PDGF-BB-induced PDGF-Rβphosphorylation in Hs27 fibroblasts with IC₅₀ values of about 1 nM.Those results are consistent with stoichiometric titration/inhibition ofall of the PDGF in the assay (1 nM monomer).

We tested another set of aptamer constructs comprising aptamers4149-8_379 and 4867-31_192 to determine the effect of overall linkerlength and the orientation of the PDGF and VEGF aptamers. See Table 21,below. With VEGF at the 5′ end, we tested one to six Heg linkers. WithPDGF at the 5′ end, we tested two to six Heg linkers, including4149-8_401, which has three Heg linkers. One Heg linker variant was nottested in this orientation because it exhibited somewhat reduced bindingin a related variant 4149-8_318. Binding data are shown in Table 16.Most of the aptamer constructs performed well, with the exception of4149-8_408 and 4149-8_409, which showed somewhat weaker affinity forPDGF-BB. The binding affinity of the Ophthotech aptamer E10030 (Fovista)is included for comparison.

The ability of the aptamer construct 4149-8_401 to inhibit the activityof both PDGF and VEGF in vitro was tested in the receptorphosphorylation experiments as described above. Aptamer construct4149-8_401 inhibited PDGF-induced PDGFRβ phosphorylation in Hs27fibroblasts with comparable potency to that of the PDGF monomer4149-8_379 (IC₅₀ values of 2.4 nM and 1.7 nM, respectively). Similarly,aptamer construct 4149-8_401 inhibited VEGF-induced VEGFR2phosphorylation in HUVEC cells with comparable potency to that of theVEGF monomer 4867-31_192 (IC₅₀ values of 0.7 nM and 2.1 nM,respectively). FIG. 18 shows the results of that experiment. FIG. 18Ashows (A) inhibition of PDGF-induced PDGF Rβ phosphorylation in Hs27fibroblasts with PDGF aptamer 4149-8_379 (open circles) and PDGF/VEGFaptamer construct 4149-8_401 (closed circles), and (B) inhibition ofVEGF-induced VEGF R2 phosphorylation in HUVECs with VEGF aptamer4867-31_192 (open circles) and PDGF/VEGF aptamer construct 4149-8_401(closed circles).

Heterodimers Based on PDGF Aptamer 5169-4 and VEGF Aptamer 4867-31.

We have designed and tested additional heterodimer constructs based onthe variants of PDGF aptamer 5169-4_26 and VEGF aptamer 4867-31_192.Aptamer constructs were synthesized head-to-tail, connected by one tosix hexaethylene glycol (Heg) linkers, in both orientations (either withthe PDGF aptamer at the 5′ end or VEGF aptamer at the 5′ end). Theresults are shown in Table 22. With VEGF at the 5′ end Heg linkersbetween three and six resulted in the highest affinities. The affinitiesin general were slightly lower when the VEGF-121 aptamer sequence was onthe 3′ end, with most K_(d) values falling in the 100-300 pM range,except for the five Heg linker sequence which had a K_(d) of 56 pM. WithPDGF at the 5′ end, the K_(d) values ranged from 11 pM for three Heglinkers to 0.54 pM for four Heg linkers, with the remaining K_(d) valuesfalling in between for all other Heg linker lengths. When PDGF was atthe 3′ end there was a trend towards higher binding affinity as thelinker length increased, with one Heg linker having a K_(d) of 5.3 pMand six Heg linkers having a K_(d) of 0.20 pM. In Table 22, “P” denotesNap-dU, superscript “1” denotes 2′-O-methyl substitution, no superscriptdenotes deoxyribonucleotides and “H” denotes hexaethylene glycol (Heg)linker.

Example 12. Simultaneous Binding of PDGF/VEGF Aptamer Constructs to VEGFand PDGF

To demonstrate the ability of the PDGF/VEGF aptamer constructs to bindVEGF and PDGF simultaneously, a sandwich assay was developed. Briefly,Nunc Maxisorp® plates were coated with either human PDGF-BB or humanVEGF-121 (20 ng/mL). After blocking the wells with a 1% BSA solution,PDGF/VEGF aptamer construct was added (10 nM) and allowed to bind to theadsorbed protein target. After washing, the biotinylated complementaryprotein (2 nM PDGF-BB for VEGF-121 coated plates and 2 nM VEGF-121 orVEGF-165 for PDGF-BB coated plates) was allowed to bind to form aternary complex. Following another wash, horseradish peroxidaseconjugated streptavidin (HRP-SA) was added and allowed to form aquaternary complex. After a final wash, a color forming horse radishperoxidase substrate was added according to the manufacturer'sdirections (Thermo Scientific TMB substrate kit 34021) and the reactionstopped when appropriate by addition of 1.6 M H₂SO₄. The absorbance perwell at 450 nm was determined with the Spectramax M5 plate reader withauto check on. In parallel to the method described above, a set of fourcontrol experiments were executed in which one of the 4 components thatmake up the quaternary complex was excluded.

As shown in FIG. 19, PDGF/VEGF aptamer construct SL1012 (20 kDaPEG-N-4149-8_401) was able to bind simultaneously to human VEGF-121 andPDGF-BB. A strong signal was observed when all components of thequaternary complex were added (complete) while the absence of any one ofthe 4 components resulted in background or near background signal.Similar results were obtained with PDGF and VEGF coated plates,indicating that the order of protein addition to the aptamer constructdid not matter. As shown in FIG. 19, SL1012 was able to bindsimultaneously to human VEGF-165 and PDGF-BB. FIG. 19A shows microtiterplates coated with VEGF with addition of biotinylated PDGF. FIG. 19Bshows microtiter plates coated with PDGF with addition of biotinylatedVEGF. Data are presented as the mean+95% confidence interval (n=3). Astrong signal was observed when all components of the quaternary complexwere added (complete) while the exclusion of any of the four componentsresulted in background or near background signal. The data alsodemonstrate that the addition of a PEG moiety to the 5′-terminus of theaptamer construct does not preclude simultaneous binding activity.

Simultaneous binding of human VEGF-165 and human PDGF-BB to (A) SL1012or and (B) SL1013 (40 kDA PEG-N-4149-8-401) is shown in FIG. 20.Microtiter plates were coated with PDGF with addition of biotinylatedVEGF. Complete means addition of all components of the quaternarycomplex, while each condition without one of the four components isshown in the graphs. Data are presented as the mean+95% confidenceinterval (n=3)

A similar experiment was performed with various aptamer constructs thatdid not contain the PEG moiety. In this experiment, only a no aptamercontrol was included because the previous results demonstrated therequirement for the other components of the complex to generate signal.As shown in FIG. 21, aptamer constructs 4149-8_317, 4149-8_318,4149-8_320, 4149-8_401 and 4149-8_414 simultaneously bound to PDGF andVEGF regardless of the order of protein addition. FIG. 21(A) showsmicrotiter plates coated with VEGF with addition of biotinylated PDGFand FIG. 21(B) shows microtiter plates coated with PDGF with addition ofbiotinylated PDGF.

Add data on simultaneous binding of heterodimer constructs based on thevariants of PDGF aptamer 5169-4 and VEGF aptamer 4867-31.

Example 13. Intravitreal Pharmacokinetic Studies

Initial ocular pharmacokinetic testing was performed to understand howthe aptamers and aptamer constructs behave in the eye. Four aptamers andaptamer constructs were tested as shown in Table 23.

For each aptamer or aptamer construct, a single intravitreal injectionwas performed into both eyes of five New Zealand White rabbits (10eyes). Animals received either a 0.5 mg/eye dose (SL1010 and SL011) or a1.0 mg/eye dose (SL1012 and SL1013). These doses represent the weight ofthe aptamer or aptamer construct only (PEG weight was excluded from thecalculations). All test articles were formulated in phosphate bufferedsaline. For each aptamer or aptamer construct test article, vitreoushumor samples were collected from both eyes from one animal at 2, 24,48, 96 or 192 hours post dose. Vitreous humor samples were stored frozenuntil they were assayed.

The vitreous humor concentrations of the aptamers or aptamer constructswere determined by ultra performance liquid chromatography (UPLC) assaymethods with detection by absorbance at 260 nanometers (nm). Briefly,the vitreous hydrogel was sheered by passing it several times through a20 gauge needle. Vitreous proteins were precipitated by the addition of2 volumes of 2-ethoxyethanol. Following centrifugation, the supernatantwas recovered and injected onto an Acquity® C18 column (0.2×100 mm). Thecolumn temperature was 80° C. and the flow rate was maintained at 0.2mL/min. Buffer A consisted of TEAA pH 7.0 and 5% acetonitrile. Buffer Bconsisted of 100% acetonitrile. The program held 50% buffer B for 1minute following the injection of sample and then buffer B was increasedlinearly to 70% over 4 minutes. Detection was accomplished by absorbanceat 260 nm. Concentrations (free acid equivalent) of aptamer or aptamerconstruct in the vitreous humor were determined by interpolation of thepeak absorbance units of the unknown samples to those obtained by astandard curve prepared with known concentrations of aptamer or aptamerconstruct.

The results of that experiment are shown in Table 24.

An ordinary linear regression fit of the natural logarithm of thevitreous concentration versus time resulted in estimates for thevitreous half-lives of 105, 47, 69 and 92 hours for SL1010, SL1011,SL1012, and SL1013, respectively. Table 25 shows the results, along withthe 95% confidence interval.

These vitreous half-lives compare favorably to the half-lives in NZWrabbits of similar sized therapeutic VEGF inhibitors, such as Macugen(83 hours, Eyetech Study Group (2002) Retina 22:143) and Lucentis (70hours, Gaudreault et al. (2007) Retina 27:1260). Therefore, theseaptamers and aptamer constructs may be useful for the treatment ofocular diseases such as AMD and diabetic retinopathy.

The foregoing embodiments and examples are intended only as examples. Noparticular embodiment, example, or element of a particular embodiment orexample is to be construed as a critical, required, or essential elementor feature of any of the claims. Various alterations, modifications,substitutions, and other variations can be made to the disclosedembodiments without departing from the scope of the present invention,which is defined by the appended claims. The specification, includingthe figures and examples, is to be regarded in an illustrative manner,rather than a restrictive one, and all such modifications andsubstitutions are intended to be included within the scope of theinvention. Accordingly, the scope of the invention should be determinedby the appended claims and their legal equivalents, rather than by theexamples given above. For example, steps recited in any of the method orprocess claims may be executed in any feasible order and are not limitedto an order presented in any of the embodiments, the examples, or theclaims.

TABLE 1Sequences Representative of the 4149-8_1 and Truncated Variants with K_(d)values for PDGF of 10 nM or less Aptamer ID. No. Sequence (5′→ 3′)SEQ ID NO. 4149-8_1C-G-C-C-C-T-C-G-T-C-C-C-A-T-C-T-C-Bn-Bn-A-C-G-A-C-Bn-A- 1C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn-A-Bn-A-G-C-G-Bn-C-A-A-C-C-C-G-A-G-Bn-G-A-A-C-A-C-C-A-A-C-C-G-A-G-A-A-C-G 4149-8_2A-T-C-T-C-Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C- 2G-Bn-Bn-Bn-A-Bn-A-G-C-G-Bn-C-A-A-C-C-C-G-A-G-Bn-G-A- A-C-A 4149-8_4A-T-C-T-C-Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C- 3G-Bn-Bn-Bn-A-Bn-A-G-C-G-Bn-C-A-A-C-C-C-G-A-G-Bn-G-A-A 4149-8_5A-T-C-T-C-Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C- 4G-Bn-Bn-Bn-A-Bn-A-G-C-G-Bn-C-A-A-C-C-C-G-A-G-Bn-G 4149-8_6A-T-C-T-C-Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C- 5G-Bn-Bn-Bn-A-Bn-A-G-C-G-Bn-C-A-A-C-C-C-G-A-G 4149-8_7A-T-C-T-C-Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C- 6G-Bn-Bn-Bn-A-Bn-A-G-C-G-Bn-C-A-A-C-C-C-G 4149-8_8A-T-C-T-C-Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C- 7G-Bn-Bn-Bn-A-Bn-A-G-C-G-Bn-C-A-A-C-C 4149-8_9A-T-C-T-C-Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C- 8G-Bn-Bn-Bn-A-Bn-A-G-C-G-Bn-C-A-A 4149-8_10A-T-C-T-C-Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C- 9G-Bn-Bn-Bn-A-Bn-A-G-C-G-Bn-C 4149-8_11C-T-C-Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn- 10Bn-Bn-A-Bn-A-G-C-G-Bn-C-A-A-C-C-C-G-A-G-Bn-G-A-A-C-A 4149-8_12C-Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 11Bn-A-Bn-A-G-C-G-Bn-C-A-A-C-C-C-G-A-G-Bn-G-A-A-C-A 4149-8_13Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn-A- 12Bn-A-G-C-G-Bn-C-A-A-C-C-C-G-A-G-Bn-G-A-A-C-A 4149-8_20C-Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 13Bn-A-Bn-A-G-C-G-Bn-C-A-A-C-C-C-G-A-G-Bn-G-A-A-C 4149-8_21C-Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 14Bn-A-Bn-A-G-C-G-Bn-C-A-A-C-C-C-G-A-G-Bn-G-A-A 4149-8_22C-Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 15Bn-A-Bn-A-G-C-G-Bn-C-A-A-C-C-C-G-A-G-Bn-G-A 4149-8_23C-Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 16Bn-A-Bn-A-G-C-G-Bn-C-A-A-C-C-C-G-A-G-Bn-G 4149-8_24C-Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 17Bn-A-Bn-A-G-C-G-Bn-C-A-A-C-C-C-G-A-G-T 4149-8_25C-Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 18Bn-A-Bn-A-G-C-G-Bn-C-A-A-C-C-C-G-A-G 4149-8_26C-Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 19Bn-A-Bn-A-G-C-G-Bn-C-A-A-C-C-C-G-A 4149-8_27C-T-C-Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn- 20Bn-Bn-A-Bn-A-G-C-G-Bn-C 4149-8_28C-Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 21Bn-A-Bn-A-G-C-G-Bn-C 4149-8_29C-Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 22Bn-A-Bn-A-G-C-G-T 4149-8_30C-Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 23 Bn-A-Bn-A-G-C-G4149-8_31 C-Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 24Bn-A-Bn-A-G-C 4149-8_32C-Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 25 Bn-A-Bn-A-G4149-8_36 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 26Bn-A-Bn-A-G-C-G-Bn-C 4149-8_37Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 27 Bn-A-Bn-A-G-C-G-T4149-8_38 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 28Bn-A-Bn-A-G-C-G 4149-8_39Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 29 Bn-A-Bn-A-G-C4149-8_40 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 30Bn-A-Bn-A-G 4149-8_44Bn-Bn-A¹-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 31 Bn-A-Bn-A-G-C-G4149-8_45 Bn-Bn-A-C¹-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 32Bn-A-Bn-A-G-C-G 4149-8_46Bn-Bn-A-C-G¹-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 33 Bn-A-Bn-A-G-C-G4149-8_47 Bn-Bn-A-C-G-A¹-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 34Bn-A-Bn-A-G-C-G 4149-8_48Bn-Bn-A-C-G-A-C¹-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 35 Bn-A-Bn-A-G-C-G4149-8_49 Bn-Bn-A-C-G-A-C-Bn-A¹-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 36Bn-A-Bn-A-G-C-G 4149-8_50Bn-Bn-A-C-G-A-C-Bn-A-C¹-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 37 Bn-A-Bn-A-G-C-G4149-8_51 Bn-Bn-A-C-G-A-C-Bn-A-C-G¹-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 38Bn-A-Bn-A-G-C-G 4149-8_52Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A¹-C-A-C-G-C-G-Bn-Bn- 39 Bn-A-Bn-A-G-C-G4149-8_53 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C¹-A-C-G-C-G-Bn-Bn- 40Bn-A-Bn-A-G-C-G 4149-8_54Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn- 41 Bn-A-Bn-A-G-C-G4149-8_55 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C¹G-C-G-Bn-Bn- 42Bn-A-Bn-A-G-C-G 4149-8_56Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G¹-C-G-Bn-Bn- 43 Bn-A-Bn-A-G-C-G4149-8_57 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C¹-G-Bn-Bn- 44Bn-A-Bn-A-G-C-G 4149-8_58Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G¹-Bn-Bn- 45 Bn-A-Bn-A-G-C-G4149-8_59 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 46Bn-A¹-Bn-A-G-C-G 4149-8_60Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 47 Bn-A-Bn-A¹-G-C-G4149-8_61 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 48Bn-A-Bn-A-G¹-C-G 4149-8_62Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 49 Bn-A-Bn-A-G-C¹-G4149-8_63 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 50Bn-A-Bn-A-G-C-G¹ 4149-8_64C3-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn- 51 A-Bn-A-G-C-G4149-8_67 Bn-Bn-A-C3-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 52Bn-A-Bn-A-G-C-G 4149-8_68Bn-Bn-A-C-C3-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 53 Bn-A-Bn-A-G-C-G4149-8_69 Bn-Bn-A-C-G-C3-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 54Bn-A-Bn-A-G-C-G 4149-8_70Bn-Bn-A-C-G-A-C3-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 55 Bn-A-Bn-A-G-C-G4149-8_71 Bn-Bn-A-C-G-A-C-C3-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn- 56A-Bn-A-G-C-G 4149-8_72Bn-Bn-A-C-G-A-C-Bn-C3-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 57 Bn-A-Bn-A-G-C-G4149-8_73 Bn-Bn-A-C-G-A-C-Bn-A-C3-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 58Bn-A-Bn-A-G-C-G 4149-8_78Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C3-A-C-G-C-G-Bn-Bn- 59 Bn-A-Bn-A-G-C-G4149-8_79 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-C3-C-G-C-G-Bn-Bn- 60Bn-A-Bn-A-G-C-G 4149-8_80Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C3-G-C-G-Bn-Bn- 61 Bn-A-Bn-A-G-C-G4149-8_87 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 62Bn-C3-Bn-A-G-C-G 4149-8_91Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 63 Bn-A-Bn-A-G-C3-G4149-8_92 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 64Bn-A-Bn-A-G-C-C3 4149-8_93Bn-Bn-A¹-C-G-A¹-C-Bn-A¹-C-G-Bn-Bn-A-C-A¹-C-G¹-C-G-Bn-Bn- 65Bn-A¹-Bn-A¹-G¹-C¹-G¹ 4149-8_94Bn-Bn-A¹-C-G-A-C-Bn-A¹-C-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn- 66Bn-A-Bn-A¹-G-C-G¹ 4149-8_95Bn-Bn-A-C-Heg-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn-A-Bn-A- 67 G-C-G 4149-8_96Bn-Bn-A-C-Heg-Heg-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn-A- 68 Bn-A-G-C-G4149-8_97 Bn-Bn-A-C-G-Heg-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn-A- 69Bn-A-G-C-G 4149-8_98Bn-Bn-A-C-C3-C3-C3-C3-C3-C3-G-Bn-Bn-A-C-A-C-G-C-G-Bn- 70Bn-Bn-A-Bn-A-G-C-G 4149-8_99Bn-Bn-A-C-C3-C3-C3-C3-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn- 71 A-Bn-A-G-C-G4149-8_100 Bn-Bn-A-C-C3-C3-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn-A-Bn- 72A-G-C-G 4149-8_101 Bn-Bn-A¹-C-G-C3-C-C3-C3-C-G-Bn-Bn-A-C-A¹-C-G¹-C-G-Bn-73 Bn-Bn-A¹-Bn-A¹-G¹-C¹-G¹ 4149-8_103U¹-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn- 74 A-Bn-A-G-C-G4149-8_105 Bn-Bn-A-C-G-A-C-U¹-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn- 75A-Bn-A-G-C-G 4149-8_106Bn-Bn-A-C-G-A-C-Bn-A-C-G--Bn-A-C-A-C-G-C-G-Bn-Bn-Bn- 76 A-Bn-A-G-C-G4149-8_110 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-U¹- 77A-Bn-A-G-C-G 4149-8_112Bn-Nap-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 78 Bn-A-Bn-A-G-C-G4149-8_113 Bn-Bn-A-C-G-A-C-Nap-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 79Bn-A-Bn-A-G-C-G 4149-8_114Bn-Bn-A-C-G-A-C-Bn-A-C-G-Nap-Bn-A-C-A-C-G-C-G-Bn-Bn- 80 Bn-A-Bn-A-G-C-G4149-8_115 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Nap-A-C-A-C-G-C-G-Bn-Bn- 81Bn-A-Bn-A-G-C-G 4149-8_116Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Nap-Bn- 82 Bn-A-Bn-A-G-C-G4149-8_117 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Nap- 83Bn-A-Bn-A-G-C-G 4149-8_118Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 84 Nap-A-Bn-A-G-C-G4149-8_119 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 85Bn-A-Nap-A-G-C-G 4149-8_121Nap-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 86 Bn-A-Bn-A-G-C-G4149-8_122 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 87Bn-A-U¹-A-G-C-G 4149-8_123Bn-Bn-A¹-C-Heg-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A- 88 Bn-A¹-G-C-G¹4149-8_124 Bn-Bn-A-C-Heg-Heg-G-Bn-Bn-A-C-A-C-G-C-G-Nap-Bn-Bn-A- 89Bn-A-G-C-G 4149-8_125Bn-Bn-A-C-Heg-Heg-G-U¹-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn-A- 90 Bn-A-G-C-G4149-8_126 Bn-Bn-A¹-C-Heg-Heg-G-U¹-Bn-A-C-A¹-C-G-C-G-Nap-Bn-Bn-A- 91Bn-A¹-G-C-G¹ 4149-8_128Bn-Nap-A¹-C-Heg-Heg-G-Nap-Nap-A-C-A¹-C-G-C-G-Nap- 92Nap-Bn-A-Nap-A¹-G-C-G¹ 4149-8_130Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 93 A¹-G-C-G¹4149-8_131 Bn-Bn-A-C-Heg-G-Bn-Bn-A-C-A-C-G-C-G-Nap-Bn-Bn-A-Bn-A- 94G-C-G 4149-8_132 Bn-Bn-A-C-Heg-G-U¹-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn-A-Bn-A- 95G-C-G 4149-8_133 Bn-Bn-A¹-C-Heg-G-U¹-Bn-A-C-A¹-C-G-C-G-Nap-Bn-Bn-A-Bn-96 A¹-G-C-G¹ 4149-8_135Pe-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹- 97 G-C-G¹4149-8_136 Bn-Pe-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹- 98G-C-G¹ 4149-8_137Bn-Bn-A¹-C-Heg-G-Pe-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹- 99 G-C-G¹4149-8_138 Bn-Bn-A¹-C-Heg-G-Bn-Pe-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹- 100G-C-G¹ 4149-8_139Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Pe-Bn-Bn-A-Bn-A¹- 101 G-C-G¹4149-8_140 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Pe-Bn-A-Bn-A¹- 102G-C-G¹ 4149-8_141Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Pe-A-Bn-A¹- 103 G-C-G¹4149-8_142 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Pe-A¹- 104G-C-G¹ 4149-8_143BT-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹- 105 G-C-G¹4149-8_144 Bn-BT-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹- 106G-C-G¹ 4149-8_145Bn-Bn-A¹-C-Heg-G-BT-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹- 107 G-C-G¹4149-8_146 Bn-Bn-A¹-C-Heg-G-Bn-BT-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹- 108G-C-G¹ 4149-8_147Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-BT-Bn-Bn-A-Bn-A¹- 109 G-C-G¹4149-8_148 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-BT-Bn-A-Bn-A¹- 110G-C-G¹ 4149-8_149Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-BT-A-Bn-A¹- 111 G-C-G¹4149-8_150 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-BT-A¹- 112G-C-G¹ 4149-8_151Th-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹- 113 G-C-G¹4149-8_152 Bn-Th-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹- 114G-C-G¹ 4149-8_153Bn-Bn-A¹-C-Heg-G-Th-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹- 115 G-C-G¹4149-8_154 Bn-Bn-A¹-C-Heg-G-Bn-Th-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹- 116G-C-G¹ 4149-8_155Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Th-Bn-Bn-A-Bn-A¹- 117 G-C-G¹4149-8_156 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Th-Bn-A-Bn-A¹- 118G-C-G¹ 4149-8_157Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Th-A-Bn-A¹- 119 G-C-G¹4149-8_158 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Th-A¹- 120G-C-G¹ 4149-8_159 Nap-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn-121 A¹-G-C-G¹ 4149-8_160Bn-Nap-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 122 A¹-G-C-G¹4149-8_161 Bn-Bn-A¹-C-Heg-G-Nap-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 123A¹-G-C-G¹ 4149-8_162Bn-Bn-A¹-C-Heg-G-Bn-Nap-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 124 A¹-G-C-G¹4149-8_163 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Nap-Bn-Bn-A-Bn- 125A¹-G-C-G¹ 4149-8_164Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Nap-Bn-A-Bn- 126 A¹-G-C-G¹4149-8_165 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Nap-A-Bn- 127A¹-G-C-G¹ 4149-8_166Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Nap- 128 A¹-G-C-G¹4149-8_167 Ib-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹- 129G-C-G¹ 4149-8_168Bn-Ib-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹- 130 G-C-G¹4149-8_169 Bn-Bn-A¹-C-Heg-G-Ib-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹- 131G-C-G¹ 4149-8_170Bn-Bn-A¹-C-Heg-G-Bn-Ib-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹- 132 G-C-G¹4149-8_171 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Ib-Bn-Bn-A-Bn-A¹- 133G-C-G¹ 4149-8_172Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Ib-Bn-A-Bn-A¹- 134 G-C-G¹4149-8_173 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Ib-A-Bn-A¹- 135G-C-G¹ 4149-8_174Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Ib-A¹- 136 G-C-G¹4149-8_175 Trp-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 137A¹-G-C-G¹ 4149-8_176Bn-Trp-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 138 A¹-G-C-G¹4149-8_177 Bn-Bn-A¹-C-Heg-G-Trp-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 139A¹-G-C-G¹ 4149-8_181Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Trp-A-Bn- 140 A¹-G-C-G¹4149-8_182 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Trp- 141A¹-G-C-G¹ 4149-8_1832Nap-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 142 A¹-G-C-G¹4149-8_184 Bn-2Nap-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 143A¹-G-C-G¹ 4149-8_185Bn-Bn-A¹-C-Heg-G-2Nap-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 144 A¹-G-C-G¹4149-8_187 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-2Nap-Bn-Bn-A-Bn- 145A¹-G-C-G¹ 4149-8_188Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-2Nap-Bn-A-Bn- 146 A¹-G-C-G¹4149-8_189 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-2Nap-A-Bn- 147A¹-G-C-G¹ 4149-8_190Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-2Nap- 148 A¹-G-C-G¹4149-8_191 2NE-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 149A¹-G-C-G¹ 4149-8_192Bn-2NE-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 150 A¹-G-C-G¹4149-8_193 Bn-Bn-A¹-C-Heg-G-2NE-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 151A¹-G-C-G¹ 4149-8_194Bn-Bn-A¹-C-Heg-G-Bn-2NE-A-C-A¹C-G-C-G-Bn-Bn-Bn-A-Bn- 152 A¹-G-C-G¹4149-8_195 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-2NE-Bn-Bn-A-Bn- 153A¹-G-C-G¹ 4149-8_197Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-2NE-A-Bn- 154 A¹-G-C-G¹4149-8_198 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-2NE- 155A¹-G-C-G¹ 4149-8_199NE-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 156 A¹-G-C-G¹4149-8_200 Bn-NE-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 157A¹-G-C-G¹ 4149-8_201Bn-Bn-A¹-C-Heg-G-NE-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 158 A¹-G-C-G¹4149-8_202 Bn-Bn-A¹-C-Heg-G-Bn-NE-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 159A¹-G-C-G¹ 4149-8_203Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-NE-Bn-Bn-A-Bn- 160 A¹-G-C-G¹4149-8_204 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-NE-Bn-A-Bn- 161A¹-G-C-G¹ 4149-8_205Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-NE-A-Bn- 162 A¹-G-C-G¹4149-8_206 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-NE- 163A¹-G-C-G¹ 4149-8_207MBn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 164 A¹-G-C-G¹4149-8_208 Bn-MBn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 165A¹-G-C-G¹ 4149-8_209Bn-Bn-A¹-C-Heg-G-MBn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 166 A¹-G-C-G¹4149-8_210 Bn-Bn-A¹-C-Heg-G-Bn-MBn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 167A¹-G-C-G¹ 4149-8_211Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-MBn-Bn-Bn-A-Bn- 168 A¹-G-C-G¹4149-8_212 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-MBn-Bn-A-Bn- 169A¹-G-C-G¹ 4149-8_213Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-MBn-A-Bn- 170 A¹-G-C-G¹4149-8_214 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-MBn- 171A¹-G-C-G¹ 4149-8_215PP-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 172 A¹-G-C-G¹4149-8_216 Bn-PP-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 173A¹-G-C-G¹ 4149-8_217Bn-Bn-A¹-C-Heg-G-PP-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 174 A¹-G-C-G¹4149-8_218 Bn-Bn-A¹-C-Heg-G-Bn-PP-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 175A¹-G-C-G¹ 4149-8_219Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-PP-Bn-Bn-A-Bn- 176 A¹-G-C-G¹4149-8_220 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-PP-Bn-A-Bn- 177A¹-G-C-G¹ 4149-8_221Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-PP-A-Bn- 178 A¹-G-C-G¹4149-8_222 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-PP- 179A¹-G-C-G¹ 4149-8_223Tyr-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 180 A¹-G-C-G¹4149-8_224 Bn-Tyr-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 181A¹-G-C-G¹ 4149-8_225Bn-Bn-A¹-C-Heg-G-Tyr-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 182 A¹-G-C-G¹4149-8_226 Bn-Bn-A¹-C-Heg-G-Bn-Tyr-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 183A¹-G-C-G¹ 4149-8_227Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Tyr-Bn-Bn-A-Bn- 184 A¹-G-C-G¹4149-8_228 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Tyr-Bn-A-Bn- 185A¹-G-C-G¹ 4149-8_229Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Tyr-A-Bn- 186 A¹-G-C-G¹4149-8_230 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Tyr- 187A¹-G-C-G¹ 4149-8_231FBn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 188 A¹-G-C-G¹4149-8_232 Bn-FBn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 189A¹-G-C-G¹ 4149-8_233Bn-Bn-A¹-C-Heg-G-FBn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 190 A¹-G-C-G¹4149-8_234 Bn-Bn-A¹-C-Heg-G-Bn-FBn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 191A¹-G-C-G¹ 4149-8_235Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-FBn-Bn-Bn-A-Bn- 192 A¹-G-C-G¹4149-8_236 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-FBn-Bn-A-Bn- 193A¹-G-C-G¹ 4149-8_237Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-FBn-A-Bn- 194 A¹-G-C-G¹4149-8_238 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-FBn- 195A¹-G-C-G¹ 4149-8_239 Bn-Bn-A¹-C ^(o)-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 196 A¹-G-C-G¹ 4149-8_240Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C ^(o) -A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 197 A¹-G-C-G¹4149-8_241 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C ^(o) -G-C-G-Bn-Bn-Bn-A-Bn-198 A¹-G-C-G¹ 4149-8_242 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C ^(o)-G-Bn-Bn-Bn-A-Bn- 199 A¹-G-C-G¹ 4149-8_243Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 200 A¹-G-C ^(o) -G¹4149-8_245 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A¹-Bn- 201A¹-G-C-G¹ 4149-8_246Bn-Bn-A¹-C-Heg-G¹-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 202 A¹-G-C-G¹4149-8_247 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G¹-C-G-Bn-Bn-Bn-A-Bn- 203A¹-G-C-G¹ 4149-8_248Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G¹-Bn-Bn-Bn-A-Bn- 204 A¹-G-C-G¹4149-8_249 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 205A¹G¹-C-G¹ 4149-8_254Tyr-Bn-A¹-C-Heg-G-FBn-Bn-A-C-A¹-C-G-C-G-2Nap-Bn-Bn-A- 206 Bn-A¹-G-C-G¹4149-8_255 Bn-Bn-A¹-C-Heg-G-Bn-Ib-A-C-A¹-C-G-C-G-Bn-Pe-Th-A-Bn-A¹- 207G-C-G¹ 4149-8_256 Bn-Bn-A¹-C-Heg-G-2Nap-Ib-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn-208 A¹-G-C-G¹ 4149-8_257Bn-Bn-A¹-C-Heg-G-NE-Ib-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹- 209 G-C-G¹4149-8_259 Bn-Bn-A¹-C-Heg-G-Ib-Ib-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹- 210G-C-G¹ 4149-8_260Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Pe-Th-A-Bn-A¹- 211 G-C-G¹4149-8_261 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Pe-Pe-A-Bn-A¹- 212G-C-G¹ 4149-8_262Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Th-Th-A-Bn-A¹- 213 G-C-G¹4149-8_263 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-2Nap-Pe-Th-A-Bn- 214A¹-G-C-G¹ 4149-8_264Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Th-Pe-Th-A-Bn-A¹- 215 G-C-G¹4149-8_265 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Ib-Pe-Th-A-Bn-A¹- 216G-C-G¹ 4149-8_266Bn-Bn-A¹-C-Heg-G-Bn-Ib-A-C-A¹-C-G-C-G-Th-Pe-Th-A-Bn-A¹- 217 G-C-G¹4149-8_267 Bn-Ib-A¹-C-Heg-G-Ib-Ib-A-C-A¹-C-G-C-G-Ib-Bn-Bn-A-Bn-A¹-G- 218C-G¹ 4149-8_268 2Nap-2Nap-A¹-C-Heg-G-2Nap-Bn-A-C-A¹-C-G-C-G-2Nap-Bn- 219Bn-A-Bn-A¹-G-C-G¹ 4149-8_269NE-Bn-A¹-C-Heg-G-NE-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-NE- 220 A¹-G-C-G¹4149-8_270 Th-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Th-Bn-Th-A-Bn-A¹- 221G-C-G¹ 4149-8_271 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A¹-Bn-222 A¹G¹-C-G¹ 4149-8_272Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A¹-Bn- 223 A¹G¹-C-G¹4149-8_273 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A¹-Bn- 224A¹-G-C-G¹ 4149-8_274Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 225 A¹-G¹-C-G¹4149-8_275 Bn-Bn-A¹-C-Heg-G-Bn-Ib-A-C-A¹-C-G-C-G-2Nap-Pe-Th-A-Bn- 226A¹-G-C-G¹ 4149-8_279Bn-Bn-A¹-C-Heg-G-Bn-Ib-A-C-A¹-C-G-C-G-Bn-Pe-Th-A-Bn-A¹- 227 G-C-G¹4149-8_280 Bn-Bn-A¹-C-Heg-G-Bn-Ib-A-C-A¹-C-G-C-G-Bn-Pe-Th-A¹-Bn-A¹- 228G-C-G¹ 4149-8_281Bn-Bn-A¹-C-Heg-G-Bn-Ib-A-C-A¹-C-G-C-G-Bn-Pe-Th-A-Bn-A¹- 229 G¹-C-G¹4149-8_282 Bn-Bn-A¹-C-Heg-G-Bn-Ib-A-C-A¹-C-G-C-G-Bn-Pe-Th-A¹-Bn- 230A¹-G-C-G¹ 4149-8_283Bn-Bn-A¹-C-Heg-G-Bn-Ib-A-C-A¹-C-G-C-G-Bn-Pe-Th-A¹-Bn- 231 A¹G¹-C-G¹4149-8_284 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C ^(o) -G-C-G-Bn-Bn-Bn-A¹-Bn-232 A¹-G-C-G¹ 4149-8_285Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C¹-A¹-C¹-G-C-G-Bn-Bn-Bn-A¹-Bn- 233 A¹-G-C¹-G¹4149-8_286 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C¹-A¹-C¹-G-C-G-Bn-Bn-Bn-A¹-Bn- 234A¹-G¹-C¹-G¹ 4149-8_287Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-C3-C3-G-C-G-Bn-Bn-Bn-A¹-Bn- 235 A¹-G-C-G¹4149-8_288 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-C3--G-C-G-Bn-Bn-Bn-A¹-Bn-A¹- 236G-C-G¹ 4149-8_289 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-C3-C¹-G-C-G-Bn-Bn-Bn-A¹-Bn-237 A¹-G-C-G¹ 4149-8_290Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C3-G-C-G-Bn-Bn-Bn-A¹-Bn- 238 A¹-G-C-G¹4149-8_291 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-Heg-G-C-G-Bn-Bn-Bn-A¹-Bn- 239A¹-G-C-G¹ 4149-8_292Bn-Bn-A-C-G-C-A-C-G-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn- 240 A-Bn-A-G-C-G4149-8_293 Bn-Bn-A-C-C-A-C-G--G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn-A- 241Bn-A-G-C-G 4149-8_294Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-T-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹- 242 G-C-G¹4149-8_295 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-C-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹- 243G-C-G¹ 4149-8_296 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-G-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹-244 G-C-G¹ 4149-8_297Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-T-G-C-G-Bn-Bn-Bn-A-Bn-A¹- 245 G-C-G¹4149-8_298 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-A-G-C-G-Bn-Bn-Bn-A-Bn- 246A¹-G-C-G¹ 4149-8_299Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-G-G-C-G-Bn-Bn-Bn-A-Bn- 247 A¹-G-C-G¹4149-8_300 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-T-G-C-G-Bn-Bn-Bn-A-Bn-A¹-G- 248C-G¹ 4149-8_301 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A-G-C-G-Bn-Bn-Bn-A-Bn-A¹-G-249 C-G¹ 4149-8_302Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-G-G-C-G-Bn-Bn-Bn-A-Bn-A¹-G- 250 C-G¹4149-8_303 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹G- 251C-G¹ 4149-8_307 Bn-Bn-A¹-C-Heg-G-Bn¹-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn-252 A¹-G-C-G¹ 4149-8_308Bn-Bn-A¹-C-Heg-G-Bn-Bn¹-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 253 A¹-G-C-G¹4149-8_311 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn¹-A-Bn- 254A¹-G-C-G¹ 4149-8_312Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn¹- 255 A¹-G-C-G¹4149-8_321 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Pe-Bn-A-MBn- 256A¹-G-C-G¹ 4149-8_322Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Pe-Th-A-MBn- 257 A¹-G-C-G¹4149-8_323 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C¹-G-C-G-Bn-Pe-Th-A¹-Bn- 258A¹-G-C-G¹ 4149-8_324Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C¹-G-C-G-Th-Pe-Th-A¹-Bn- 259 A¹-G-C-G¹4149-8_325 Bn-Bn-A¹-C-Heg-G-Bn¹-Bn-A-C-A¹-C¹-G-C-G-Bn-Bn-Bn-A¹-Bn- 260A¹-G-C-G¹ 4149-8_326Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C¹-G-C-G-Bn-Bn-Bn¹-A¹-Bn- 261 A¹-G-C-G¹4149-8_327 Bn-Bn-A¹-C-Heg-G-Bn¹-Bn-A-C-A¹-C¹-G-C-G-Bn-Bn-Bn¹-A¹-Bn- 262A¹-G-C-G¹ 4149-8_328Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C¹-G-C-G-Bn-Pe-Bn¹-A¹Bn- 263 A¹-G-C-G¹4149-8_329 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C¹-G-C-G-Bn-Bn-Bn-A¹- 264MBn-A¹-G-C-G¹ 4149-8_330Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C¹-G-C-G-Bn-Pe-Bn-A¹- 265 MBn-A¹-G-C-G¹4149-8_331 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C¹-G-C-G-Bn-Pe-Th-A¹- 266MBn-A¹-G-C-G¹ 4149-8_332Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-C3-C¹-G-C-G-Bn-Pe-Bn-A¹- 267 MBn-A¹-G-C-G¹4149-8_333 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-C3-C¹-G-C-G-Bn-Pe-Th-A¹- 268MBn-A¹-G-C-G¹ 4149-8_334Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Pe-Th-A-Bn-A¹- 269G-C-G¹-Heg-Heg-Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Pe-Th-A-Bn-A¹-G-C-G¹ 4149-8_335Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Pe-Th-A-Bn-A¹- 270G-C-G¹-Heg-Heg-Heg-Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Pe-Th-A-Bn-A¹-G-C-G¹ 4149-8_336Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Pe-Th-A-Bn-A¹- 271G-C-G¹-Heg-Heg-Heg-Heg-Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Pe-Th-A-Bn-A¹-G-C-G¹ 4149-8_337Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Pe-Th-A-Bn-A¹- 272G-C-G¹-Heg-Heg-Heg-Heg-Heg-Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Pe-Th-A-Bn-A¹-G-C-G¹ 4149-8_338Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Pe-Th-A-Bn-A¹- 273G-C-G¹-Heg-Heg-Heg-Heg-Heg-Heg-Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Pe-Th-A-Bn-A¹-G-C-G¹ 4149-8_339Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Pe-Th-A-Bn-A¹- 274G-C-G¹-C3-(3′-Doubler)-T 4149-8_340Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Pe-Th-A-Bn-A¹- 275G-C-G¹-Heg-(3′-Doubler)-T 4149-8_341Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Pe-Th-A-Bn-A¹- 276G-C-G¹-Heg-Heg-(3′-Doubler)-T 4149-8_342Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Pe-Th-A-Bn-A¹- 277G-C-G¹-Heg-Heg-Heg-(3′-Doubler)-T 4149-8_343Bn-Bn-A¹-C-Heg-G-Bn¹-Bn-A-C-A¹-C¹-G-C-G-Bn-Pe-Bn¹-A¹-Bn- 278 A¹-G-C-G¹4149-8_344 Bn-Bn-A¹-C-Heg-Gg-Bn¹-Bn-A-C-A¹-C¹-G-C-G-Bn-Pe-Bn¹-A¹- 279Bn-A¹-G-C-G¹ 4149-8_345 Bn-Bn-A¹-C-Heg-G-Bn¹-Bn-A-C-A¹-C¹-G ^(o)-C-G-Bn-Pe-Bn¹-A¹- 280 Bn-A¹-G-C-G¹ 4149-8_346Bn-Bn-A¹-C-Heg-G-Bn¹-Bn-A-C-A¹-C¹-G-C-G ^(o) -Bn-Pe-Bn¹-A¹- 281Bn-A¹-G-C-G¹ 4149-8_347Bn-Bn-A¹-C-Heg-G-Bn¹-Bn-A-C-A¹-C¹-G-C-G-Bn-Pe-Bn¹-A¹Bn- 282 A¹-G ^(o)-C-G¹ 4149-8_349 Bn-Bn-A¹-C ^(o)-Heg-G-Bn¹-Bn-A-C-A¹-C¹-G-C-G-Bn-Pe-Bn¹-A¹- 283 Bn-A¹-G-C-G¹ 4149-8_352Bn-Bn-A¹-C-Heg-G-Bn¹-Bn-A-C-A¹-C¹-G-C-G-Bn-Pe-Bn¹-A¹Bn- 284 A¹-G-C ^(o)-G¹ 4149-8_353 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A ^(o)-C-A¹-C¹-G-C-G-Bn-Pe-Bn¹-A¹- 285 Bn-A¹-G-C-G¹ 4149-8_3542Nap-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C¹-G-C-G-Bn-Pe-Bn¹-A¹- 286 Bn-A¹-G-C-G¹4149-8_355 FBn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C¹-G-C-G-Bn-Pe-Bn¹-A¹- 287Bn-A¹-G-C-G¹ 4149-8_356NE-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C¹-G-C-G-Bn-Pe-Bn¹-A¹-Bn- 288 A¹-G-C-G¹4149-8_357 2NE-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C¹-G-C-G-Bn-Pe-Bn¹-A¹- 289Bn-A¹-G-C-G¹ 4149-8_358PP-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C¹-G-C-G-Bn-Pe-Bn¹-A¹-Bn- 290 A¹-G-C-G¹4149-8_359 Ib-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C¹-G-C-G-Bn-Pe-Bn¹-A¹-Bn- 291A¹-G-C-G¹ 4149-8_360Bn-MBn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C¹-G-C-G-Bn-Pe-Bn¹-A¹- 292 Bn-A¹-G-C-G¹4149-8_361 T-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹- 293G-C-G¹ 4149-8_362 Bn-T-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹-294 G-C-G¹ 4149-8_363Bn-Bn-A¹-C-Heg-G-T-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹- 295 G-C-G¹4149-8_369 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A²-C-A¹-C¹-G-C-G-Bn-Pe-Bn¹-A¹-Bn- 296A¹-G-C-G¹ 4149-8_370Bn-Bn-A¹-C-Heg-G-Bn¹-Bn-A²-C-A¹-C¹-G-C-G-Bn-Pe-Bn¹-A¹- 297 Bn-A¹-G-C-G¹4149-8_371 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C¹-G-C-G²-Bn-Pe-Bn¹-A¹-Bn- 298A¹-G-C-G¹ 4149-8_372Bn-Bn-A¹-C-Heg-G-Bn¹-Bn-A-C-A¹-C¹-G-C-G²-Bn-Pe-Bn¹-A¹- 299 Bn-A¹-G-C-G¹4149-8_373 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A²-C-A¹-C¹-G-C-G²-Bn-Pe-Bn¹-A¹- 300Bn-A¹-G-C-G¹ 4149-8_374Bn-Bn-A¹-C-Heg-G-Bn¹-Bn-A²-C-A¹-C¹-G-C-G²-Bn-Pe-Bn-A¹- 301 Bn-A¹-G-C-G¹4149-8_375 FBn-Bn-A¹-C-Heg-G-Bn¹-Bn-A-C-A¹-C¹-G-C-G-Bn-Bn-Bn¹-A¹- 302Bn-A¹-G-C-G¹ 4149-8_376Bn-MBn-A¹-C-Heg-G-Bn¹-Bn-A-C-A¹-C¹-G-C-G-Bn-Bn-Bn¹-A¹- 303 Bn-A¹-G-C-G¹4149-8_377 FBn-MBn-A¹-C-Heg-G-Bn¹-Bn-A-C-A¹-C¹-G-C-G-Bn-Bn-Bn¹-A¹- 304Bn-A¹-G-C-G¹ 4149-8_378FBn-Bn-A¹-C-Heg-G-Bn¹-Bn-A-C-C3-C¹-G-C-G-Bn-Bn-Bn¹-A¹- 305 Bn-A¹-G-C-G¹4149-8_379 Bn-MBn-A¹-C-Heg-G-Bn¹-Bn-A-C-C3-C¹-G-C-G-Bn-Bn-Bn¹-A¹- 306Bn-A¹-G-C-G¹ 4149-8_380FBn-MBn-A¹-C-Heg-G-Bn¹-Bn-A-C-C3-C¹-G-C-G-Bn-Bn-Bn¹- 307 A¹-Bn-A¹-G-C-G¹4149-8_381 FBn-Bn-A¹-C-Heg-G-Bn¹-Bn-A-C-A¹-C¹-G-C-G-Bn-Pe-Bn¹-A¹- 308Bn-A¹-G-C-G¹ 4149-8_382Bn-MBn-A¹-C-Heg-G-Bn¹-Bn-A-C-A¹-C¹-G-C-G-Bn-Pe-Bn¹-A¹- 309 Bn-A¹-G-C-G¹4149-8_383 FBn-MBn-A¹-C-Heg-G-Bn¹-Bn-A-C-A¹-C¹-G-C-G-Bn-Pe-Bn¹-A¹- 310Bn-A¹-G-C-G¹ 4149-8_384FBn-Bn-A¹-C-Heg-G-Bn¹-Bn-A-C-C3-C¹-G-C-G-Bn-Pe-Bn¹-A¹- 311 Bn-A¹-G-C-G¹4149-8_385 Bn-MBn-A¹-C-Heg-G-Bn¹-Bn-A-C-C3-C¹-G-C-G-Bn-Pe-Bn¹-A¹- 312Bn-A¹-G-C-G¹ 4149-8_386FBn-MBn-A¹-C-Heg-G-Bn¹-Bn-A-C-C3-C¹-G-C-G-Bn-Pe-Bn¹-A¹- 313 Bn-A¹-G-C-G¹4149-8_388 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A²-C-A¹-C¹-G-C-G²-Bn-Bn-Bn¹-A¹- 314Bn-A¹-G-C-G¹ 4149-8_389Bn-Bn-A¹-C-Heg-G-Bn¹-Bn-A²-C-A¹-C¹-G-C-G²-Bn-Bn-Bn¹-A¹- 315 Bn-A¹-G-C-G¹4149-8_390 Bn-Bn-A¹-C-Heg-G-Bn¹-Bn-A²-C-C3-C¹-G-C-G²-Bn-Pe-Bn¹-A¹- 316Bn-A¹-G-C-G¹ 4149-8_391Bn-Bn-A¹-C-Heg-G-Bn-Bn-A²-C-C3-C¹-G-C-G²-Bn-Bn-Bn¹-A¹- 317 Bn-A¹-G-C-G¹4149-8_392 Bn-Bn-A¹-C-Heg-G-Bn¹-Bn-A²-C-C3-C¹-G-C-G²-Bn-Bn-Bn¹-A¹- 318Bn-A¹-G-C-G¹ 4149-8_393FBn-Bn-A¹-C-Heg-G-Bn-Bn-A²-C-A¹-C¹-G-C-G²-Bn-Pe-Bn¹-A¹- 319 Bn-A¹-G-C-G¹4149-8_394 Bn-MBn-A¹-C-Heg-G-Bn-Bn-A²-C-A¹-C¹-G-C-G²-Bn-Pe-Bn¹-A¹- 320Bn-A¹-G-C-G¹ 4149-8_395FBn-MBn-A¹-C-Heg-G-Bn-Bn-A²-C-A¹-C¹-G-C-G²-Bn-Pe-Bn¹- 321A¹-Bn-A¹-G-C-G¹ 4149-8_396FBn-Bn-A¹-C-Heg-G-Bn-Bn-A²-C-C3-C¹-G-C-G²-Bn-Pe-Bn¹-A¹- 322 Bn-A¹-G-C-G¹4149-8_397 Bn-MBn-A¹-C-Heg-G-Bn-Bn-A²-C-C3-C¹-G-C-G²-Bn-Pe-Bn¹-A¹- 323Bn-A¹-G-C-G¹ 4149-8_398FBn-MBn-A¹-C-Heg-G-Bn-Bn-A²-C-C3-C¹-G-C-G²-Bn-Pe-Bn¹- 324A¹-Bn-A¹-G-C-G¹ 4149-8_399FBn-MBn-A¹-C-Heg-G-Bn¹-Bn-A²-C-C3-C¹-G-C-G²-Bn-Bn-Bn¹- 325A¹-Bn-A¹-G-C-G¹ 4149-8_400FBn-MBn-A¹-C-Heg-G-Bn¹-Bn-A²-C-A¹-C¹-G-C-G²-Bn-Bn-Bn¹- 326A¹-Bn-A¹-G-C-G¹ 4149-8_402Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-G-C-G--Bn-Bn-Bn- 327 A-Bn-A-G-C4149-8_403 Bn-Bn-A-C-Heg-G-Bn-Bn-A-C-A-G-C-G--Bn-Bn-Bn-A-Bn-A-G- 328 C4149-8_404 Bn-Bn-A-C-Heg-G-Bn-Bn-A-C-C3-G-C-G-Bn-Bn-Bn-A-Bn-A-G- 329 C4149-8_418 Bn-MBn-A¹-C-Heg-G-Bn¹-Bn-A-C-C3-C¹-G-C-G¹-Bn-Bn-Bn¹-A¹- 330Bn-A¹-G-C-G¹ 4149-8_419Bn-MBn-A¹-C-Heg-G-Bn¹-Bn-A-C-C3-C¹-G-C-G¹-Nap-Bn-Bn¹- 331A¹-Bn-A¹-G-C-G¹ 4149-8_420Bn-MBn-A¹-C-Heg-G-Bn¹-Bn-A-C-C3-C¹-G-C-G¹-2Nap-Bn-Bn¹- 332A¹-Bn-A¹-G-C-G¹ 4149-8_421Bn-MBn-A¹-C-Heg-G-Bn¹-Bn-A-C-C3-C¹-G¹-C-G-Bn-Bn-Bn¹-A¹- 333 Bn-A¹-G-C-G¹4149-8_422 Bn-MBn-A¹-C-Heg-G-Bn¹-Bn-A-C-C3-C¹-G¹-C-G¹-Bn-Bn-Bn¹- 334A¹-Bn-A¹-G-C-G¹ 4149-8_423Bn-MBn-A¹-C-Heg-G-Bn¹-Bn-A-C-C3-C¹-G¹-C-G-Nap-Bn-Bn¹- 335A¹-Bn-A¹-G-C-G¹ 4149-8_424Bn-MBn-A¹-C-Heg-G-Bn¹-Bn-A-C-C3-C¹-G¹-C-G-Bn-Bn-Bn¹- 336 A¹-Bn-A¹-G-C-G¹4149-8_425 Bn-MBn-A¹-C-Heg-G-Bn¹-Bn-A¹-C-C3-C¹-G-C-G-Bn-Bn-Bn¹-A¹- 337Bn-A¹-G-C-G¹ 4149-8_431Bn-MBn-A¹-C-Heg-G-Bn¹-Bn-A²-C-C3-C¹-G-C-G²-Nap-Bn2- 338Bn¹-A¹-Bn-A¹-G-C-G¹ 4149-8_432Bn-MBn-A¹-C-Heg-G-Bn¹-Bn-A²-C-C3-C¹-G-C-G²-Bn-Bn2-Bn¹- 339A¹-Bn-A¹-G-C-G¹ 4149-8_433Bn-MBn-A¹-C-Heg-G-Bn¹-Bn-A²-C-C3-C¹-G-C-G²-Nap-Bn-Bn¹- 340A¹-Bn-A¹-G-C-G¹ 4149-8_434Bn-MBn-A¹-C-Heg-G-Bn¹-Bn-A²-C-C3-C¹-G-C-G²-Bn-Bn-Bn¹- 341A¹-Bn-A¹-G-C-G¹ 4149-8_435Bn-Bn-A¹-C-Heg-G-Bn¹-Bn-A²-C-C3-C¹-G-C-G²-Nap-Bn2-Bn¹- 342A¹-Bn-A¹-G-C-G¹ 4149-8_436Bn-Bn-A¹-C-Heg-G-Bn¹-Bn-A²-C-C3-C¹-G-C-G²-Bn-Bn2-Bn¹-A¹- 343Bn-A¹-G-C-G¹ 4149-8_437Bn-Bn-A¹-C-Heg-G-Bn¹-Bn-A²-C-C3-C¹-G-C-G²-Nap-Bn-Bn¹-A¹- 344Bn-A¹-G-C-G¹ No superscript indicates deoxyribose Superscript oindicates 2′-fluoro Superscript 1 indicates 2′-O-methyl Superscript 2indicates phosphorothioate (deoxyribose) C3 = three carbon linker Heg =hexaethylene glycol linker Nap = naphthyl-dU Pe = phenethyl-dU BT =benzothiophenyl-dU Th = thiophenyl-dU Ib = isobutyl-dU Trp =tryptaminyl-dU 2Nap = 2-naphthyl-dU 2NE = 2-naphthylethyl-dU NE =naphthylethyl-dU MBn = methylenedioxybenzyl-dU PP = phenpropyl-dU Tyr =tyrosyl-dU FBn = fluorobenzyl-dU Bn = benzyl-dU 3′-Doubler = SymmetricDoubler Phosphoramidite (Glen Research, Cat# 10-1920-02)

TABLE 1a Homodimers of PDGF aptamer 4149-8_260 (SL5): SOMAmer (2.5 nM)PDGF-BB (1 nM) duplicates Ratio to Relative PDGFRb SeqI Kd (M) 4149-8_38phosphorylation 4149-8_38 3.00E−11 1.0 5.4% 3.6% 4149-8_334 4.92E−11 1.61.7% 1.7% 4149-8_335 5.46E−11 1.8 1.5% 1.5% 4149-8_336 1.84E−11 0.611.7% 2.3% 4149-8_337 3.79E−12 0.1 4149-8_338 2.84E−11 0.93 1.4% 1.3%4149-8_339 6.20E−12 0.2 2.7% 3.6% 3′DBLR 4149-8_340 1.89E−11 0.6 1.7%3.2% 3′DBLR 4149-8_341 2.72E−11 0.9 1.5% 1.9% 3′DBLR 4149-8_342 1.81E−110.6 1.6% 1.5% 3′DBLR

TABLE 2 Sequences Representative of the Truncated Variants with K_(d) values for PDGFof more than 10 nM Aptamer SEQ ID. No. Sequence (5′→3′) ID NO. 4149-8_14C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn-A-Bn-A- 345G-C-G-Bn-C-A-A-C-C-C-G-A-G-Bn-G-A-A-C-A 4149-8_15A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn-A-Bn-A-G-C- 346G-Bn-C-A-A-C-C-C-G-A-G-Bn-G-A-A-C-A 4149-8_16Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn-A-Bn-A-G-C-G- 347Bn-C-A-A-C-C-C-G-A-G-Bn-G-A-A-C-A 4149-8_17C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn-A-Bn-A-G-C-G-Bn-C-A- 348A-C-C-C-G-A-G-Bn-G-A-A-C-A 4149-8_18Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn-A-Bn-A-G-C-G-Bn-C-A-A-C- 349C-C-G-A-G-Bn-G-A-A-C-A 4149-8_19A-C-A-C-G-C-G-Bn-Bn-Bn-A-Bn-A-G-C-G-Bn-C-A-A-C-C-C-G- 350A-G-Bn-G-A-A-C-A 4149-8_33C-Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 351 Bn-A-Bn-A4149-8_34 C-Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 352Bn-A-T 4149-8_35 C-Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-353 Bn-A 4149-8_41Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn- 354 A-Bn-A4149-8_42 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn- 355 A-T4149-8_43 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn- 356 A4149-8_65 Bn-C3-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn- 357A-Bn-A-G-C-G 4149-8_66Bn-Bn-C3-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 358 Bn-A-Bn-A-G-C-G4149-8_74 Bn-Bn-A-C-G-A-C-Bn-A-C-C3-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 359Bn-A-Bn-A-G-C-G 4149-8_75Bn-Bn-A-C-G-A-C-Bn-A-C-G-C3-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn- 360 A-Bn-A-G-C-G4149-8_76 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-C3-A-C-A-C-G-C-G-Bn-Bn-Bn- 361A-Bn-A-G-C-G 4149-8_77Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-C3-C-A-C-G-C-G-Bn-Bn- 362 Bn-A-Bn-A-G-C-G4149-8_81 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-C3-C-G-Bn-Bn- 363Bn-A-Bn-A-G-C-G 4149-8_82Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C3-G-Bn-Bn- 364 Bn-A-Bn-A-G-C-G4149-8_83 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-C3-Bn-Bn- 365Bn-A-Bn-A-G-C-G 4149-8_84Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-C3-Bn-Bn- 366 A-Bn-A-G-C-G4149-8_85 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-C3-Bn- 367A-Bn-A-G-C-G 4149-8_86Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-C3- 368 A-Bn-A-G-C-G4149-8_88 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn- 369A-C3-A-G-C-G 4149-8_89Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn- 370 A-Bn-C3-G-C-G4149-8_90 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn- 371A-Bn-A-C3-C-G 4149-8_102Bn-Bn-A¹-C-G-C3-C-C3-C3-C-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn- 372Bn-A-Bn-A¹-G-C-G¹ 4149-8_104Bn-U¹-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn- 373 A-Bn-A-G-C-G4149-8_107 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-U¹-A-C-A-C-G-C-G-Bn-Bn-Bn- 374A-Bn-A-G-C-G 4149-8_108Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-U¹-Bn-Bn- 375 A-Bn-A-G-C-G4149-8_109 Bn-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-U¹-Bn- 376A-Bn-A-G-C-G 4149-8_111Nap-Bn-A-C-G-A-C-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn- 377 Bn-A-U¹-A-G-C-G4149-8_127 Bn-Nap-A-C-Heg-Heg-G-U¹-Nap-A-C-A-C-G-C-G-Nap-Nap- 378Bn-A-Nap-A-G-C-G 4149-8_129Bn-Nap-A¹-C-Heg-Heg-G-U¹-Nap-A-C-A¹-C-G-C-G-Nap-Nap- 379Bn-A-Nap-A¹-G-C-G¹ 4149-8_134Bn-Bn-A-C-G-Bn-Bn-A-C-A-C-G-C-G-Bn-Bn-Bn-A-Bn-A-G-C-G 380 4149-8_178Bn-Bn-A¹-C-Heg-G-Bn-Trp-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 381 A¹-G-C-G¹4149-8_179 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Trp-Bn-Bn-A-Bn- 382A¹-G-C-G¹ 4149-8_180Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Trp-Bn-A-Bn- 383 A¹-G-C-G¹4149-8_186 Bn-Bn-A¹-C-Heg-G-Bn-2Nap-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 384A¹-G-C-G¹ 4149-8_196Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-2NE-Bn-A-Bn- 385 A¹-G-C-G¹4149-8_244 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A¹-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 386A¹-G-C-G¹ 4149-8_250Tyr-MBn-A¹-C-Heg-G-FBn-Ib-A-C-A¹-C-G-C-G-2Nap-Pe-Th-A- 387 NE-A¹-G-C-G¹4149-8_251 Tyr-MBn-A¹-C-Heg-G-FBn-Ib-A-C-A¹-C-G-C-G-2 Na p-Pe-Th- 388A¹-NE-A¹-G¹-C-G¹ 4149-8_252Trp-Ib-A¹-C-Heg-G-NE-Ib-A-C-A¹-C-G-C-G-Th-Pe-Th-A-MBn- 389 ALG-C-G¹4149-8_253 Trp-Ib-A¹-C-Heg-G-NE-Ib-A-C-A¹-C-G-C-G-Th-Pe-Th-A¹-MBn- 390A¹G¹-C-G¹ 4149-8_258Bn-Bn-A¹-C-Heg-G-U¹-Ib-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹- 391 G-C-G¹4149-8_276 Bn-Bn-A¹-C-Heg-G-FBn-Ib-A-C-A¹-C-G-C-G-Bn-Pe-Th-A-Bn- 392ALG-C-G¹ 4149-8_277Tyr-Bn-A¹-C-Heg-G-Bn-Ib-A-C-A¹C-G-C-G-Bn-Pe-Th-A-Bn-A¹- 393 G-C-G¹4149-8_278 Tyr-Bn-A¹-C-Heg-G-FBn-Ib-A-C-A¹-C-G-C-G-2Nap-Pe-Th-A- 394Bn-A¹-G-C-G¹ 4149-8_304Bn-Bn-A¹-C-C3-G-Bn-Bn-A-C-C3--G-C-G-Bn-Bn-Bn-A-Bn-A¹- 395 G-C-G¹4149-8_305 Bn¹-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 396ALG-C-G¹ 4149-8_306Bn-Bn¹-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn- 397 ALG-C-G¹4149-8_309 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn¹-Bn-Bn-A-Bn- 398A¹-G-C-G¹ 4149-8_310Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn¹-Bn-A-Bn- 399 A¹-G-C-G¹4149-8_348 Bn-Bn-A¹-C-Heg-G-Bn¹-Bn-A-̊-C-A¹-C-G-C-G-Bn-Pe-Bn¹-A¹- 400Bn-A¹-G-C-G¹ 4149-8_350Bn-Bn-A1-C-Heg-G-Bn¹-Bn-A-C-̊-A¹-C-G-C-G-Bn-Pe-Bn¹-A¹- 401 Bn-A¹-G-C-G¹4149-8_351 Bn-Bn-A¹-C-Heg-G-Bn¹-Bn-A-C-A¹-C-G-C-̊-G-Bn-Pe-Bn¹-A¹- 402Bn-A¹-G-C-G¹ 4149-8_364Bn-Bn-A¹-C-Heg-G-Bn-T-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-Bn-A¹- 403 G-C-G¹4149-8_365 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-T-Bn-Bn-A-Bn-A¹- 404G-C-G¹ 4149-8_366 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-T-Bn-A-Bn-A¹-405 G-C-G¹ 4149-8_367Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-T-A-Bn-A¹- 406 G-C-G¹4149-8_368 Bn-Bn-A¹-C-Heg-G-Bn-Bn-A-C-A¹-C-G-C-G-Bn-Bn-Bn-A-T-A¹- 407G-C-G¹ 4149-8_405 Bn-Bn-A-C-Heg-G-Bn-Bn-A-C-C3-C-G-Bn-Bn-Bn-A-Bn-A-G 4084149-8_426 Bn-MBn-A¹-C-Heg-G-Bn¹-Bn-A¹-C-C3-C-G-C-G¹-Bn-Bn-Bn¹- 409A¹-Bn-A¹-G-C-G¹ No superscript indicates deoxyribose Superscript -indicates 2′-fluoro Superscript 1 indicates 2′-O-methyl Superscript 2indicates phosphorothioate (deoxyribose) C3 = three carbon linker Heg =hexaethylene glycol linker Nap = naphthyl-dU Pe = phenethyl-dU BT =benzothiophenyl-dU Th = thiophenyl-dU Ib = isobutyl-dU Trp =tryptaminyl-dU 2Nap = 2-naphthyl-dU 2NE = 2-naphthylethyl-dU NE =naphthylethyl-dU MB = methylenedioxybenzyl-dU PP = phenpropyl-dU Tyr =tyrosyl-dU FBn = fluorobenzyl-dU Bn = benzyl-dU

TABLE 3  Truncations of PDGF aptamer Clone 4149-8_1. Seq. Aptamer K_(d)ID. ID. No. Sequence (5′→3′) Length (nM) NO. 4149-8_2A-T-C-T-C-Z-Z-A-C-G-A-C-Z-A-C-G- 50 0.05 2Z-Z-A-C-A-C-G-C-G-Z-Z-Z-A-Z-A-G- C-G-Z-C-A-A-C-C-C-G-A-G-Z-G-A-A- C-A4149-8_5 A-T-C-T-C-Z-Z-A-C-G-A-C-Z-A-C-G- 46 0.02 4Z-Z-A-C-A-C-G-C-G-Z-Z-Z-A-Z-A-G- C-G-Z-C-A-A-C-C-C-G-A-G-Z-G 4149-8_7A-T-C-T-C-Z-Z-A-C-G-A-C-Z-A-C-G- 42 0.02 6Z-Z-A-C-A-C-G-C-G-Z-Z-Z-A-Z-A-G- C-G-Z-C-A-A-C-C-C-G 4149-8_9A-T-C-T-C-Z-Z-A-C-G-A-C-Z-A-C-G- 38 0.03 8Z-Z-A-C-A-C-G-C-G-Z-Z-Z-A-Z-A-G- C-G-Z-C-A-A 4149-8_10A-T-C-T-C-Z-Z-A-C-G-A-C-Z-A-C-G- 36 0.05 9Z-Z-A-C-A-C-G-C-G-Z-Z-Z-A-Z-A-G- C-G-Z-C 4149-8_11C-T-C-Z-Z-A-C-G-A-C-Z-A-C-G-Z-Z- 48 0.03 10A-C-A-C-G-C-G-Z-Z-Z-A-Z-A-G-C-G- Z-C-A-A-C-C-C-G-A-G-Z-G-A-A-C-A4149-8_12 C-Z-Z-A-C-G-A-C-Z-A-C-G-Z-Z-A-C- 46 0.04 11A-C-G-C-G-Z-Z-Z-A-Z-A-G-C-G-Z-C- A-A-C-C-C-G-A-G-Z-G-A-A-C-A 4149-8_13Z-A-C-G-A-C-Z-A-C-G-Z-Z-A-C-A-C- 44 0.8 12G-C-G-Z-Z-Z-A-Z-A-G-C-G-Z-C-A-A- C-C-C-G-A-G-Z-G-A-A-C-A 4149-8_14C-G-A-C-Z-A-C-G-Z-Z-A-C-A-C-G-C- 42 13 345G-Z-Z-Z-A-Z-A-G-C-G-Z-C-A-A-C-C- C-G-A-G-Z-G-A-A-C-A 4149-8_16Z-A-C-G-Z-Z-A-C-A-C-G-C-G-Z-Z-Z- 38 22 347A-Z-A-G-C-G-Z-C-A-A-C-C-C-G-A-G- Z-G-A-A-C-A 4149-8_18Z-Z-A-C-A-C-G-C-G-Z-Z-Z-A-Z-A-G- 34 13 349C-G-Z-C-A-A-C-C-C-G-A-G-Z-G-A-A- C-A 4149-8_19A-C-A-C-G-C-G-Z-Z-Z-A-Z-A-G-C-G- 32 74 350Z-C-A-A-C-C-C-G-A-G-Z-G-A-A-C-A 4149-8_26C-Z-Z-A-C-G-A-C-Z-A-C-G-Z-Z-A-C- 39 0.08 19A-C-G-C-G-Z-Z-Z-A-Z-A-G-C-G-Z-C- A-A-C-C-C-G-A 4149-8_27C-T-C-Z-Z-A-C-G-A-C-Z-A-C-G-Z-Z- 34 0.09 20A-C-A-C-G-C-G-Z-Z-Z-A-Z-A-G-C-G- Z-C 4149-8_29C-Z-Z-A-C-G-A-C-Z-A-C-G-Z-Z-A-C- 31 0.07 22A-C-G-C-G-Z-Z-Z-A-Z-A-G-C-G-T 4149-8_30 C-Z-Z-A-C-G-A-C-Z-A-C-G-Z-Z-A-C-30 0.07 23 A-C-G-C-G-Z-Z-Z-A-Z-A-G-C-G 4149-8_31C-Z-Z-A-C-G-A-C-Z-A-C-G-Z-Z-A-C- 29 0.30 24 A-C-G-C-G-Z-Z-Z-A-Z-A-G-C4149-8_32 C-Z-Z-A-C-G-A-C-Z-A-C-G-Z-Z-A-C- 28 6 25A-C-G-C-G-Z-Z-Z-A-Z-A-G 4149-8_33 C-Z-Z-A-C-G-A-C-Z-A-C-G-Z-Z-A-C-27 >1000 351 A-C-G-C-G-Z-Z-Z-A-Z-A 4149-8_37Z-Z-A-C-G-A-C-Z-A-C-G-Z-Z-A-C-A- 30 0.04 27 C-G-C-G-Z-Z-Z-A-Z-A-G-C-G-T4149-8_38 Z-Z-A-C-G-A-C-Z-A-C-G-Z-Z-A-C-A- 29 0.05 28C-G-C-G-Z-Z-Z-A-Z-A-G-C-G 4149-8_39 Z-Z-A-C-G-A-C-Z-A-C-G-Z-Z-A-C-A- 28.11 29 C-G-C-G-Z-Z-Z-A-Z-A-G-C 4149-8_40Z-Z-A-C-G-A-C-Z-A-C-G-Z-Z-A-C-A- 27 1.5 30 C-G-C-G-Z-Z-Z-A-Z-A-G4149-8_41 Z-Z-A-C-G-A-C-Z-A-C-G-Z-Z-A-C-A- 26 >1000 354C-G-C-G-Z-Z-Z-A-Z-A

TABLE 4 Data-collection, refinement and model statistics. Ligand4149-8_260 4149-8_255 DATA COLLECTION Space group P4₁2₁2 P4₁2₁2Unit-cell 59.10, 59.10, 167.01 59.40, 59.40 168.2 parameters: a, b, c(Å) Wavelength (Å) 0.97918 0.97918 Resolution range 50.0-2.20(2.26-2.20) 50.0-2.30 (2.36-2.30) (Å) Unique reflections 15629 13750Completeness (%) 96.8 (99.1) 97.2 (98.4) R_(merge) 4.0 (57.8) 6.4 (57.2)Mean I/σ(I) 25.3 (3.4) 19.3 (2.6) REFINEMENT Resolution range 50.0-2.20(2.26-2.20) 50.0-2.30 (2.36-2.30) (Å) R_(cryst) 0.225 (0.329) 0.239(0.366) R_(free) 0.262 (0.353) 0.279 (0.526) R.m.s.d. bonds (Å) 0.0180.018 R.m.s.d. angles (°) 1.882 2.017 Total atoms 1382 1355 Mean Bfactor (Å²) 43.68 43.86 Residues in favored 98.9 98.9 region (%)Residues in allowed 100.0 100.0 region (%) Molprobity score 1.45(99^(th)) 1.41 (99^(th)) (percentile) PDB ID 4HQU 4HQX Values inparenthesis indicate the values for the highest of twenty resolutionshells R_(merge) = Σ_(h)Σ_(i)|I_(i)(h) − <I(h)>|/Σ_(h)Σ_(i)I_(i)(h)R_(free) = Σ_(h)||F_(obs)| − |F_(calc)||/Σ_(h)|F_(obs)|. The free Rfactor was calculated using 5% of the reflections omitted from therefinement (The CCP4 suite: programs for protein crystallography, 1994).*Ligand B-factors are for ligands in the active sites of the proteinmonomers. Ligands from solvent (PEG, glycerol, etc.) were not includedin the calculation.

TABLE 5 Base-pair parameters for the PDGF BB aptamer compared to B-formDNA. Parameter B-DNA 5′Stem loop Miniknot S1 Miniknot S2 Complementarybase-pair parameters Buckle (deg.) 0.5 _((6.7)) −5.67 _((7.90)) 0.32_((22.8)) −0.74 _((15.3)) Propeller (deg.) −11.4 _((5.3)) −9.84_((6.10)) 7.43 _((10.4)) 2.42 _((7.75)) Opening (deg.) 0.60 _((3.1))−8.13 _((25.2)) 7.86 _((12.5)) 0.35 _((0.40)) Shear (Å) 0.00 _((0.21))1.12 _((1.42)) 1.29 _((2.52)) −0.03 _((0.42)) Stretch (Å) −0.15_((0.12)) −0.38 _((0.66)) 1.96 _((3.55)) −0.12 _((0.42)) Stagger (Å)0.09 _((0.19)) 0.04 _((0.61)) −0.45 _((0.69)) 0.28 _((0.05)) U2, A3/ A3,C4/ C10, A9/ A9, U20/ C12, G13/ G13, C14/ Base-pair step parameters U7,U8 G6, U7 U16, G15 U17, U16 C23, G24 G22, C23 Tilt (deg.) −0.1 _((2.5))−0.80 −1.45 −4.65 −5.83 −2.39 3.86 Roll (deg.) 0.6 _((5.2)) 0.34 0.556.12 −5.64 4.19 3.94 Twist (deg.) 36 _((6.8)) 24.1 33.3 25.3 15.9 2933.9 Shift (Å) −0.02 _((0.45)) 3.20 −0.12 −0.90 −1.45 −0.17 0.78 Slide(Å) 0.23 _((0.81)) 0.25 −0.24 0.27 −4.49 0.21 −0.4 Rise (Å) 3.32_((0.19)) 2.68 3.28 2.98 3.58 2.73 3.13 Local helical parametersInclination (deg.) 2.1 _((9.2)) 0.81 0.96 13.6 −18.9 8.28 6.72 Tip(deg.) 0.0 _((4.3)) 1.92 2.52 10.4 19.5 4.72 −6.57 Helical twist (deg.)36.5 _((6.6)) 24.1 33.4 26.3 17.8 29.4 34.3 x-displacement (Å) 0.05_((1.28)) 0.52 −0.5 −0.97 −9.84 −0.34 −1.28 y-displacement (Å) 0.02_((0.87)) −7.89 −0.03 0.81 −0.12 −0.09 −0.73 Helical rise (Å) 3.29_((0.21)) 2.58 3.28 3.07 5.08 2.73 3.13

TABLE 6  Truncations of PDGF aptamer clone 5169-4. Aptamer K_(d) Seq.ID. No. Sequence (5′→3′) (nM) ID. NO. 5169-4_3C-T-G-C-C-P-C-G-A-C-A-G-C-A-P-G-P-A- 0.029 410P-G-C-A-C-A-P-C-P-A-G-A-G-G-C-P-A-C- C-C-A-G-P-G-A-A-P-G-A-G-G-A5169-4_4 C-C-P-C-G-A-C-A-G-C-A-P-G-P-A-P-G-C- 0.027 411A-C-A-P-C-P-A-G-A-G-G-C-P-A-C-C-C-A- G-P-G-A-A-P-G-A-G-G-A 5169-4_5C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- 0.031 412P-C-P-A-G-A-G-G-C-P-A-C-C-C-A-G-P-G- A-A-P-G-A-G-G-A 5169-4_6C-A-G-C-A-P-G-P-A-P-G-C-A-C-A-P-C-P- 67 413A-G-A-G-G-C-P-A-C-C-C-A-G-P-G-A-A-P- G-A-G-G-A 5169-4_7C-T-G-C-C-P-C-G-A-C-A-G-C-A-P-G-P-A- 0.014 414P-G-C-A-C-A-P-C-P-A-G-A-G-G-C-P-A-C- C-C-A-G-P-G-A-A-P-G-A 5169-4_8C-T-G-C-C-P-C-G-A-C-A-G-C-A-P-G-P-A- 0.036 415P-G-C-A-C-A-P-C-P-A-G-A-G-G-C-P-A-C- C-C-A-G-P-G-A-A 5169-4_9C-T-G-C-C-P-C-G-A-C-A-G-C-A-P-G-P-A- 0.72 416P-G-C-A-C-A-P-C-P-A-G-A-G-G-C-P-A-C- C-C-A-G-P 5169-4_10C-T-G-C-C-P-C-G-A-C-A-G-C-A-P-G-P-A- 0.63 417P-G-C-A-C-A-P-C-P-A-G-A-G-G-C-P-A-C- C-C 5169-4_11C-T-G-C-C-P-C-G-A-C-A-G-C-A-P-G-P-A- 0.60 418P-G-C-A-C-A-P-C-P-A-G-A-G-G-C-P-A 5169-4_12C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- 0.064 419P-C-P-A-G-A-G-G-C-P-A-C-C-C-A-G-P-G- A-A 5169-4_13G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A-P- 0.027 420C-P-A-G-A-G-G-C-P-A-C-C-C-A-G-P-G-A- A 5169-4_14A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A-P-C- 0.17 421P-A-G-A-G-G-C-P-A-C-C-C-A-G-P-G-A-A 5169-4_15C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- 0.019 422P-C-P-A-G-A-G-G-C-P-A-C-C-C-A-G-P-G- A 5169-4_16C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- 0.016 423P-C-P-A-G-A-G-G-C-P-A-C-C-C-A-G-P-G 5169-4_17C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- 0.020 424P-C-P-A-G-A-G-G-C-P-A-C-C-C-A-G-P 5169-4_18C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- 0.026 425 P-C-P-A-G-A-G-G-C-P-A5169-4_19 C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- 0.012 426P-C-P-A-G-A-G-G-C-P 5169-4_20 C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- 0.014427 P-C-P-A-G-A-G-G-C 5169-4_21 C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A-0.021 428 P-C-P-A-G-A-G-G 5169-4_22 C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A-0.015 429 P-C-P-A-G-A-G 5169-4_23 C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A-0.020 430 P-C-P-A-G-A 5169-4_24 C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A-0.021 431 P-C-P-A-G 5169-4_25 C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- 0.020432 P-C-P-A 5169-4_26 C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- 0.017 433P-C-P 5169-4_27 G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A-P- 0.062 434 C-P-A-G-A5169-4_28 G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A-P- 0.048 435 C-P-A-G5169-4_29 G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A-P- 0.062 436 C-P-A 5169-4_30G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A-P- 0.062 437 C-P 5169-4_32G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A-P- 0.053 438 C-P-A-G-A-G

TABLE 7  C3 spacer substitutions at all positions in 5169-4_26.Aptamer ID. K_(d) Seq. ID. No. Sequence (5′→3′) (nM) NO. 5169-4_59C3-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C- 0.053 439 A-P-C-P 5169-4_60C-C3-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C- 27 440 A-P-C-P 5169-4_61C-G-C3-C-A-G-C-A-P-G-P-A-P-G-C-A-C- NB 441 A-P-C-P 5169-4_62C-G-A-C3-A-G-C-A-P-G-P-A-P-G-C-A-C- 42 442 A-P-C-P 5169-4_63C-G-A-C-C3-G-C-A-P-G-P-A-P-G-C-A-C- 29 443 A-P-C-P 5169-4_64C-G-A-C-A-C3-C-A-P-G-P-A-P-G-C-A-C- 0.054 444 A-P-C-P 5169-4_65C-G-A-C-A-G-C3-A-P-G-P-A-P-G-C-A-C- 0.074 445 A-P-C-P 5169-4_66C-G-A-C-A-G-C-C3-P-G-P-A-P-G-C-A-C- 8.5 446 A-P-C-P 5169-4_67C-G-A-C-A-G-C-A-C3-G-P-A-P-G-C-A-C- NB 447 A-P-C-P 5169-4_68C-G-A-C-A-G-C-A-P-C3-P-A-P-G-C-A-C- NB 448 A-P-C-P 5169-4_69C-G-A-C-A-G-C-A-P-G-C3-A-P-G-C-A-C- NB 449 A-P-C-P 5169-4_70C-G-A-C-A-G-C-A-P-G-P-C3-P-G-C-A-C- NB 450 A-P-C-P 5169-4_71C-G-A-C-A-G-C-A-P-G-P-A-C3-G-C-A-C- NB 451 A-P-C-P 5169-4_72C-G-A-C-A-G-C-A-P-G-P-A-P-C3-C-A-C- 230 452 A-P-C-P 5169-4_73C-G-A-C-A-G-C-A-P-G-P-A-P-G-C3-A-C- 0.015 453 A-P-C-P 5169-4_74C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-C3-C- 0.37 454 A-P-C-P 5169-4_75C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C3- 0.14 455 A-P-C-P 5169-4_76C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C- 1.3 456 C3-P-C-P 5169-4_77C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- NB 457 C3-C-P 5169-4_78C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- 0.28 458 P-C3-P 5169-4_79C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- NB 459 P-C-C3

TABLE 8  2′O-methyl and deoxythymidine substitutions in 5169-4_26.Aptamer ID. K_(d) Seq. ID. No. Sequence (5′→3′) (nM) NO. 5169-4_33C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C- 0.027 460 A-P-C-P 5169-4_34C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C- 0.033 461 A-P-C-P 5169-4_35C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C- 0.016 462 A-P-C-P 5169-4_36C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C- 0.028 463 A-P-C-P 5169-4_37C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C- 0.041 464 A-P-C-P 5169-4_38C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- 0.022 465 P-C-P 5169-4_39C-G¹-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C- 0.058 466 A-P-C-P 5169-4_40C-G-A-C-A-G¹-C-A-P-G-P-A-P-G-C-A-C- 0.024 467 A-P-C-P 5169-4_41C-G-A-C-A-G-C-A-P-G¹-P-A-P-G-C-A-C- 0.097 468 A-P-C-P 5169-4_42C-G-A-C-A-G-C-A-P-G-P-A-P-G¹-C-A-C- 0.0069 469 A-P-C-P 5169-4_43C-G-A¹-C-A-G-C-A-P-G-P-A-P-G-C-A-C- 0.16 470 A-P-C-P 5169-4_44C-G-A-C-A¹-G-C-A-P-G-P-A-P-G-C-A-C- 0.53 471 A-P-C-P 5169-4_45C-G-A-C-A-G-C-A¹-P-G-P-A-P-G-C-A-C- 0.93 472 A-P-C-P 5169-4_46C-G-A-C-A-G-C-A-P-G-P-A¹-P-G-C-A-C- 0.093 473 A-P-C-P 5169-4_47C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A¹-C- 0.016 474 A-P-C-P 5169-4_48C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C- 0.014 475 A¹-P-C-P 5169-4_49C-G-A-C-A-G-C-A-P¹-G-P-A-P-G-C-A-C- 0.53 476 A-P-C-P 5169-4_50C-G-A-C-A-G-C-A-P-G-P¹-A-P-G-C-A-C- 0.047 477 A-P-C-P 5169-4_51C-G-A-C-A-G-C-A-P-G-P-A-P¹-G-C-A-C- 0.32 478 A-P-C-P 5169-4_52C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- 0.31 479 P¹-C-P 5169-4_53C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- 0.022 480 P-C-P¹ 5169-4_54C-G-A-C-A-G-C-A-T-G-P-A-P-G-C-A-C-A- 44 481 P-C-P 5169-4_55C-G-A-C-A-G-C-A-P-G-T-A-P-G-C-A-C-A- 7.8 482 P-C-P 5169-4_56C-G-A-C-A-G-C-A-P-G-P-A-T-G-C-A-C-A- NB 483 P-C-P 5169-4_57C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- 120 484 T-C-P 5169-4_58C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- NB 485 P-C-T

TABLE 9 Multiple 2′-O-methyl substitutions in PDGF aptamer 5169-4_26.Aptamer ID. Seq. ID. No. Sequence (5′→3′) K_(d) (nM) NO. 5169 4_80C G A C A G C¹ A P G P A P G C A¹ C- 0.0061 486 A¹ P C P 5169 4_81C G A C A G C¹ A P G P A P G¹ C A¹ C- 0.0049 487 A¹ P C P 5169 4_82C G A C A G¹ C¹ A P G P A P G¹ C A¹- 0.0016 488 C A¹ P C¹ P¹ 5169 4_83C G A C A G¹ C¹ A P G P A P G¹ C¹ A¹- 0.0021 489 C A¹ P C¹ P¹ 5169 4_84C  G A C¹ A G¹ C¹ A P G P A P G¹ C¹- 0.0053 490 A¹ C A¹ P C¹ P¹5169 4_85 C¹ G A C¹ A G¹ C¹ A P G P¹ A P G¹ C¹- 0.0062 491A¹ C¹ A¹ P C¹ P¹ 5169 4_86 C¹ G¹ A C¹ A G¹ C¹ A P G P¹ A P G¹ C¹- 0.064492 A¹ C¹ A¹ P C¹ P¹ 5169 4_87 C¹ G¹ A C¹ A G¹ C¹ A P G¹ P¹ A¹ P G¹- NB493 C¹ A¹ C¹ A¹ P C¹ P¹ 5169 4_88 C¹ G¹ A¹ C¹ A G¹ C¹ A P G¹ P¹ A¹ P G¹-NB 494 C¹ A¹ C¹ A¹ P C¹ P¹ 5169 4_89C¹ G¹ A¹ C¹ A G¹ C¹ A P G¹ P¹ A¹ P¹ G¹- NB 495 C¹ A¹ C¹ A¹ P¹ C¹ P¹5169 4_90 C¹ G¹ A¹ C¹ A¹ G¹ C¹ A P¹ G¹ P¹ A¹ P¹- NB 496G¹ C¹ A¹-C¹ A¹ P¹ C¹ P¹ 5169 4_91C¹ G  A¹ C¹ A¹ G¹ C¹ A¹ P¹ G¹ P¹ A¹ P¹- NB 497 G¹ C¹ A¹ C¹ A¹ P¹ C¹ P¹5169 4_92 C¹ G A C A G C¹ A P G P A P G C¹ A¹- 0.0052 498 C A¹ P C¹ P¹5169 4_105 C G A C A G¹ C A P G P A P G C A C- 0.0061 499 A¹ P C¹ P5169-4_106 C¹-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C- 0.016 517 A¹-P-C¹-P5169-4_107 C¹-G-A-C-A-G¹-C-A-P-G-P-A-P-G-C-A-C- 0.013 518 A¹-P-C-P5169-4_108 C¹-G-A-C-A-G¹-C-A-P-G-P-A-P-G¹-C-A-C- 0.012 519 A-P-C-P5169-4_109 C¹-G-A-C-A-G¹-C-A-P-G-P-A-P-G-C-A¹-C- 0.011 520 A-P-C-P5169-4_110 C¹-G-A-C-A-G-C-A-P-G-P-A-P-G¹-C-A¹-C- 0.0050 521 A-P-C-P5169-4_111 C-G-A-C-A-G-C¹-A-P-G-P-A-P-G¹-C-A¹-C- 0.0048 522 A-P-C-P5169-4_112 C-G-A-C-A-G-C-A-P-G-P-A-P-G¹-C-A¹-C- 0.0062 523 A-P-C-P¹5169-4_113 C-G-A-C-A-G-C¹-A-P-G-P-A-P-G-C-A¹-C- 0.012 524 A-P-C-P¹5169-4_114 C-G-A-C-A-G-C¹-A-P-G-P-A-P-G-C-A-C¹- 0.038 525 A-P-C-P¹5169-4_115 C-G-A-C¹-A-G-C¹-A-P-G-P-A-P-G-C-A-C- 0.024 526 A-P-C-P¹5169-4_116 C-G-A-C¹-A-G-C-A-P-G-P-A-P-G-C-A-C¹- 0.093 527 A-P-C-P¹5169-4_117 C-G-A-C¹-A-G-C-A-P-G-P-A¹-P-G-C-A-C¹- 0.71 528 A-P-C-P5169-4_118 C-G¹-A-C¹-A-G-C-A-P-G-P-A-P-G-C-A-C¹- 0.56 529 A-P-C-P5169-4_119 C-G¹-A-C¹-A-G-C-A-P-G-P-A-P-G-C-A-C¹- NB 530 A-P-C-P5169-4_120 C-G¹-A-C-A-G-C-A-P-G-P-A¹-P-G-C¹-A-C- NB 531 A-P-C-P5169-4_121 C-G¹-A-C-A-G-C-A-P-G¹-P-A¹-P-G-C-A-C- NB 532 A-P-C-P5169-4_122 C-G¹-A-C-A-G-C-A-P-G¹-P-A-P-G-C¹-A-C- NB 533 A-P-C-P5169-4_123 C¹-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C- 0.030 534 A-P-C¹-P¹5169-4_124 C-G-A-C-A¹-G-C-A-P-G-P-A-P-G-C-A-C- 0.60 535 A-P-C¹-P¹5169-4_125 C¹-G-A-C-A¹-G-C-A-P-G-P-A-P-G-C-A-C- 1.0 536 A-P-C¹-P5169-4_126 C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- NB 537 P¹-C¹-P¹5169-4_127 C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A¹-C¹- 0.010 538 A¹-P-C-P5169-4_128 C-G-A-C-A-G-C-A-P-G-P-A-P¹-G¹-C¹-A-C- 0.099 539 A-P-C-P5169-4_129 C-G-A-C-A-G-C-A-P-G¹-P¹-A¹-P-G-C-A-C- NB 540 A-P-C-P5169-4_130 C-G-A-C-A-G-C¹-A¹-P¹-G-P-A-P-G-C-A-C- 4.5 541 A-P-C-P5169-4_131 C-G-A-C¹-A¹-G¹-C-A-P-G-P-A-P-G-C-A-C- 0.64 542 A-P-C-P5169-4_132 C¹-G¹-A¹-C-A-G-C-A-P-G-P-A-P-G-C-A-C- 0.42 543 A-P-C-P5169-4_144 C¹-G-A-C-A-G-C¹-A-P-G-P-A-P-G¹-C-A¹- 0.00050 544 C-A¹-P-C-P¹5169-4_146 C¹-G-A-C¹-A-G¹-C¹-A-P-G-P-A-P-G¹-C¹- 0.00060 545A¹-C¹-A¹-P-C¹-P¹

TABLE 10 Truncations of VEGF aptamer Clone 4867-31. Aptamer ID.K_(d) (nM) K_(d) (nM) K_(d) (nM) Seq. ID. No. length Sequence (5′→ 3′)hVEGF165 rVEGF164 hVEGF121 NO. OH-4867- 38 T-C-A-A-P-C-A-G-C-C-G-P-P-0.26 0.54 0.82 546 31_22 C-A-A-G-P-G-C-P-P-G-P-A-G-G-A-P-P-P-A-A-A-P-G-G-A OH-4867- 37 T-C-A-A-P-C-A-G-C-C-G-P-P- 0.30 0.661.3 547 31_23 C-A-A-G-P-G-C-P-P-G-P-A-G- G-A-P-P-P-A-A-A-P-G-G OH-4867-36 T-C-A-A-P-C-A-G-C-C-G-P-P- 2.8 4.2 36 548 31_24C-A-A-G-P-G-C-P-P-G-P-A-G- G-A-P-P-P-A-A-A-P-G OH-4867- 35T-C-A-A-P-C-A-G-C-C-G-P-P- 26 16 70 549 31_25 C-A-A-G-P-G-C-P-P-G-P-A-G-G-A-P-P-P-A-A-A-P OH-4867-31- 39 C-A-A-P-C-A-G-C-C-G-P-P-C- 0.075 0.070.49 550 26 A-A-G-P-G-C-P-P-G-P-A-G-G- A-P-P-P-A-A-A-P-G-G-A-A-POH-4867-31- 38 A-A-P-C-A-G-C-C-G-P-P-C-A- 0.11 0.07 0.59 551 27A-G-P-G-C-P-P-G-P-A-G-G-A- P-P-P-A-A-A-P-G-G-A-A-P OH-4867-31- 37A-P-C-A-G-C-C-G-P-P-C-A-A- 0.10 0.07 0.97 552 28G-P-G-C-P-P-G-P-A-G-G-A-P- P-P-A-A-A-P-G-G-A-A-P OH-4867-31- 36P-C-A-G-C-C-G-P-P-C-A-A-G- 0.13 0.08 0.96 553 29P-G-C-P-P-G-P-A-G-G-A-P-P- P-A-A-A-P-G-G-A-A-P OH-4867-31- 35C-A-G-C-C-G-P-P-C-A-A-G-P- 0.20 0.15 1.4 554 30G-C-P-P-G-P-A-G-G-A-P-P-P- A-A-A-P-G-G-A-A-P OH-4867-31- 34A-G-C-C-G-P-P-C-A-A-G-P-G- 0.17 0.12 1.0 555 31C-P-P-G-P-A-G-G-A-P-P-P-A- A-A-P-G-G-A-A-P OH-4867-31- 38C-A-A-P-C-A-G-C-C-G-P-P-C- 0.10 0.06 0.51 556 31A-A-G-P-G-C-P-P-G-P-A-G-G- A-P-P-P-A-A-A-P-G-G-A-A OH-4867-31- 37A-A-P-C-A-G-C-C-G-P-P-C-A- 0.11 0.04 0.55 557 33A-G-P-G-C-P-P-G-P-A-G-G-A- P-P-P-A-A-A-P-G-G-A-A OH-4867-31- 36A-P-C-A-G-C-C-G-P-P-C-A-A- 0.35 0.05 0.47 558 34G-P-G-C-P-P-G-P-A-G-G-A-P- P-P-A-A-A-P-G-G-A-A OH-4867-31- 35P-C-A-G-C-C-G-P-P-C-A-A-G- 0.11 0.05 flat 559 35P-G-C-P-P-G-P-A-G-G-A-P-P- P-A-A-A-P-G-G-A-A OH-4867-31- 34C-A-G-C-C-G-P-P-C-A-A-G-P- 0.11 0.06 0.39 560 36G-C-P-P-G-P-A-G-G-A-P-P-P- A-A-A-P-G-G-A-A OH-4867-31- 33A-G-C-C-G-P-P-C-A-A-G-P-G- 0.092 0.04 0.21 561 37C-P-P-G-P-A-G-G-A-P-P-P-A- A-A-P-G-G-A-A OH-4867-31- 37C-A-A-P-C-A-G-C-C-G-P-P-C- 0.17 0.09 0.81 562 38A-A-G-P-G-C-P-P-G-P-A-G-G- A-P-P-P-A-A-A-P-G-G-A OH-4867-31- 36A-A-P-C-A-G-C-C-G-P-P-C-A- 0.20 0.09 0.62 563 39A-G-P-G-C-P-P-G-P-A-G-G-A- P-P-P-A-A-A-P-G-G-A OH-4867-31- 35A-P-C-A-G-C-C-G-P-P-C-A-A- 0.14 0.10 0.70 564 40G-P-G-C-P-P-G-P-A-G-G-A-P- P-P-A-A-A-P-G-G-A OH-4867-31- 34P-C-A-G-C-C-G-P-P-C-A-A-G- 0.13 0.08 0.63 565 41P-G-C-P-P-G-P-A-G-G-A-P-P- P-A-A-A-P-G-G-A OH-4867-31- 33C-A-G-C-C-G-P-P-C-A-A-G-P- 0.071 0.05 0.20 566 42G-C-P-P-G-P-A-G-G-A-P-P-P- A-A-A-P-G-G-A OH-4867-31- 32A-G-C-C-G-P-P-C-A-A-G-P-G- 0.050 0.05 0.29 567 42C-P-P-G-P-A-G-G-A-P-P-P-A- A-A-P-G-G-A OH-4867-31- 32C-A-G-C-C-G-P-P-C-A-A-G-P- 0.13 0.38 0.14 568 44G-C-P-P-G-P-A-G-G-A-P-P-P- A-A-A-P-G-G OH-4867-31- 31C-A-G-C-C-G-P-P-C-A-A-G-P- 1.1 4.5 3.3 569 45 G-C-P-P-G-P-A-G-G-A-P-P-P-A-A-A-P-G OH-4867-31- 30 C-A-G-C-C-G-P-P-C-A-A-G-P- 5.9 9.6 NB 570 46G-C-P-P-G-P-A-G-G-A-P-P-P- A-A-A-P OH-4867-31- 29C-A-G-C-C-G-P-P-C-A-A-G-P- NB 83 NB 571 47 G-C-P-P-G-P-A-G-G-A-P-P-P-A-A-A OH-4867-31- 28 C-A-G-C-C-G-P-P-C-A-A-G-P- NB 142 NB 572 48G-C-P-P-G-P-A-G-G-A-P-P-P- A-A OH-4867-31- 27 C-A-G-C-C-G-P-P-C-A-A-G-P-NB NB NB 573 49 G-C-P-P-G-P-A-G-G-A-P-P-P-A OH-4867-31- 26C-A-G-C-C-G-P-P-C-A-A-G-P- NB NB NB 574 50 G-C-P-P-G-P-A-G-G-A-P-P-POH-4867-31- 31 A-G-C-C-G-P-P-C-A-A-G-P-G- 0.11 0.074 0.20 575 51C-P-P-G-P-A-G-G-A-P-P-P-A- A-A-P-G-G OH-4867-31- 30A-G-C-C-G-P-P-C-A-A-G-P-G- 0.17 0.58 2.8 576 52C-P-P-G-P-A-G-G-A-P-P-P-A- A-A-P-G OH-4867-31- 29A-G-C-C-G-P-P-C-A-A-G-P-G- 3.2 NB 19 577 53 C-P-P-G-P-A-G-G-A-P-P-P-A-A-A-P OH-4867-31- 28 A-G-C-C-G-P-P-C-A-A-G-P-G- NB NB NB 578 54C-P-P-G-P-A-G-G-A-P-P-P-A- A-A OH-4867-31- 27 A-G-C-C-G-P-P-C-A-A-G-P-G-NB NB NB 579 55 C-P-P-G-P-A-G-G-A-P-P-P-A-A OH-4867-31- 26A-G-C-C-G-P-P-C-A-A-G-P-G- NB NB NB 580 56 C-P-P-G-P-A-G-G-A-P-P-P-AOH-4867-31- 25 A-G-C-C-G-P-P-C-A-A-G-P-G- NB NB NB 581 57C-P-P-G-P-A-G-G-A-P-P-P OH-4867-31- 31 G-C-C-G-P-P-C-A-A-G-P-G-C- 0.100.21 0.15 582 58 P-P-G-P-A-G-G-A-P-P-P-A-A- A-P-G-G-A OH-4867-31- 30C-C-G-P-P-C-A-A-G-P-G-C-P- 0.16 0.20 0.14 583 59P-G-P-A-G-G-A-P-P-P-A-A-A- P-G-G-A OH-4867-31- 29C-G-P-P-C-A-A-G-P-G-C-P-P- 0.61 1.4 2.2 584 60G-P-A-G-G-A-P-P-P-A-A-A-P- G-G-A OH-4867-31- 28G-P-P-C-A-A-G-P-G-C-P-P-G- 2.9 13 33 585 61 P-A-G-G-A-P-P-P-A-A-A-P-G-G-A OH-4867- 29 C-C-G-P-P-C-A-A-G-P-G-C-P- 0.17 586 31_143P-G-P-A-G-G-A-P-P-P-A-A-A- P-G-G OH-4867- 30 G-C-C-G-P-P-C-A-A-G-P-G-C-0.20 587 31_144 P-P-G-P-A-G-G-A-P-P-P-A-A- A-P-G-G

TABLE 11 C3 spacer substitutions at all positions in 4867-15_2.Aptamer ID. K_(d) ratio to  Seq. ID. No. Sequence 5′→ 3′ (nM) parent NO.OH-4867-31_2 C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G-0.15 1 588 G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-V-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- 0.11 0.72 58924 G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-V-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- 0.0 0.84 59025 G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-V-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- 0.20 1.3 59126 G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-V-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A- 0.40 2.7 592 27G-G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-V-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- 0.34 2.3 59328 G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-V-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- 0.20 1.4 59429 G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-V-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- 0.11 0.76 59530 G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-V-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A- 0.095 0.64 59631 G-G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-V-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- 0.079 0.53597 32 G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-V-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- 0.12 0.83 59833 G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-V-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- 0.14 0.95 59934 G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-V-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- 3.4 23 600 35G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-V-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- 23 153 601 36G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-V-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- 30 205 602 37G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-V-P-C-A-A-G-P-G-C-P-P-G-P-A- NB — 603 38G-G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-V-C-A-A-G-P-G-C-P-P-G-P-A- NB — 604 39G-G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-V-A-A-G-P-G-C-P-P-G-P-A-G- 0.17 1.1 60540 G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-V-A-G-P-G-C-P-P-G-P-A-G- 13 85 606 41G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-V-G-P-G-C-P-P-G-P-A-G- 31 207 607 42G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-V-P-G-C-P-P-G-P-A-G- 9.2 62 608 43G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-V-G-C-P-P-G-P-A- NB — 609 44G-G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-V-C-P-P-G-P-A-G- 3.5 24 610 45G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-V-P-P-G-P-A-G- NB — 611 46G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-V-P-G-P-A- NB — 612 47G-G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-V-G-P-A- NB — 613 48G-G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-V-P-A-G- 0.079 0.54614 49 G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-V-A- 3.5 24 615 50G-G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-V-G- 1.6 11 616 51G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-V- 0.20 1.3 61752 G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- 23 157 618 53V-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- NB — 619 54G-V-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- NB — 620 55G-A-V-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- NB — 621 56G-A-P-V-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- NB — 622 57G-A-P-P-V-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- NB — 623 58G-A-P-P-P-V-A-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- NB — 624 59G-A-P-P-P-A-V-A-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- NB — 625 60G-A-P-P-P-A-A-V-P-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- NB — 626 61G-A-P-P-P-A-A-A-V-G-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- 53 361 627 62G-A-P-P-P-A-A-A-P-V-G-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- 15 101 628 63G-A-P-P-P-A-A-A-P-G-V-A-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- 0.22 1.5 62964 G-A-P-P-P-A-A-A-P-G-G-V-A-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- 0.13 0.88 63065 G-A-P-P-P-A-A-A-P-G-G-A-V-P-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- 0.11 0.72 63166 G-A-P-P-P-A-A-A-P-G-G-A-A-V-P-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- 0.15 0.99 63267 G-A-P-P-P-A-A-A-P-G-G-A-A-P-V-G-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- 0.32 2.1 63368 G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-V-G-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- 0.77 5.2 63469 G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-V-A-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- 0.68 4.6 63570 G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-V-G-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- 0.24 1.6 63671 G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-V-G-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- 0.19 1.3 63772 G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-V-A OH-4867-15-C-C-C-T-C-C-A-P-C-A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G- 0.17 1.1 63873 G-A-P-P-P-A-A-A-P-G-G-A-A-P-P-G-G-A-G-G-V

TABLE 12 2′-O-methyl substitutions in VEGF aptamer 4867-31_43 ratio Seq.K_(d) to ID. Aptamer ID. No. Sequence 5′ → 3′ (nM) parent NO.OH-4867-31-43 A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A- 0.29 1.0 567G-G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_65A¹-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A- NB — 639G-G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_66A-G¹-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A- 0.17 0.6 640G-G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_67A-G-C¹-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A- 0.33 1.2 641G-G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_68A-G-C-C¹-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A- 7.55 26.0 642G-G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_69A-G-C-C-G¹-P-P-C-A-A-G-P-G-C-P-P-G-P-A- NB — 643G-G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_70A-G-C-C-G-U¹-P-C-A-A-G-P-G-C-P-P-G-P-A- NB — 644G-G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_71A-G-C-C-G-P-U¹-C-A-A-G-P-G-C-P-P-G-P-A- NB — 645G-G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_72A-G-C-C-G-P-P-C¹-A-A-G-P-G-C-P-P-G-P-A- 0.21 0.7 646G-G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_73A-G-C-C-G-P-P-C-A¹-A-G-P-G-C-P-P-G-P-A- 5.99 20.7 647G-G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_74A-G-C-C-G-P-P-C-A-A¹-G-P-G-C-P-P-G-P-A- 3.14 10.8 648G-G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_75A-G-C-C-G-P-P-C-A-A-G¹-P-G-C-P-P-G-P-A- 0.15 0.5 649G-G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_76A-G-C-C-G-P-P-C-A-A-G-U¹-G-C-P-P-G-P-A- 22.90 79.0 650G-G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_77A-G-C-C-G-P-P-C-A-A-G-P-G¹-C-P-P-G-P-A- 6.64 22.9 651G-G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_78A-G-C-C-G-P-P-C-A-A-G-P-G-C¹-P-P-G-P-A- 0.24 0.8 652G-G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_79A-G-C-C-G-P-P-C-A-A-G-P-G-C-U¹-P-G-P-A- NB — 653G-G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_80A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-U¹-G-P-A- NB — 654G-G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_81A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G¹-P-A- 0.44 .5 655G-G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_82A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-U¹-A- NB — 656G-G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_83A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A¹- 0.10 0.3 657G-G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_84A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A- 0.21 0.7 658G¹-G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_85A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A- 25.80 89.0 659G-G¹-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_86A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A- 6.21 21.4 660G-G-A¹-P-P-P-A-A-A-P-G-G-A OH-4867-31_87A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A- NB — 661G-G-A-U¹-P-P-A-A-A-P-G-G-A OH-4867-31_88A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A- NB — 662G-G-A-P-U¹-P-A-A-A-P-G-G-A OH-4867-31_89A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A- NB — 663G-G-A-P-P-U¹-A-A-A-P-G-G-A OH-4867-31_90A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A- 2.85 9.8 664G-G-A-P-P-P-A¹-A-A-P-G-G-A OH-4867-31_91A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A- 0.35 1.2 665G-G-A-P-P-P-A-A¹-A-P-G-G-A OH-4867-31_92A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A- 8.87 30.6 666G-G-A-P-P-P-A-A-A¹-P-G-G-A OH-4867-31_93A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A- NB — 667G-G-A-P-P-P-A-A-A-U¹-G-G-A OH-4867-31_94A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A- 0.21 0.7 668G-G-A-P-P-P-A-A-A-P-G¹-G-A OH-4867-31_95A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A- 0.19 0.7 669G-G-A-P-P-P-A-A-A-P-G-G¹-A OH-4867-31_96A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A- 0.13 0.4 670G-G-A-P-P-P-A-A-A-P-G-G-A¹ OH-4867-31_97A-G-C-C-G-P-P-V-A-A-G-P-G-C-P-P-G-P-A- 7.30 25.2 671G-G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_98A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-V-P-A- 0.10 0.3 672G-G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_99A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A- 0.22 0.8 673V-G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_100A-G-C-C-G-P-P-V-A-A-G-P-G-C-P-P-V-P-A- NB — 674V-G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_101A-G-C-C-G-P-P-A-A-G-P-G-C-P-P-G-P-A-G- 5.89 20.3 675G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_102A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-P-A-G- NB — 676 G-A-P-P-P-A-A-A-P-G-G-AOH-4867-31_103 A-G-C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A- NB — 677G-A-P-P-P-A-A-A-P-G-G-A OH-4867-31_145C-C-G-P-P-A-A-A-G-P-G-C-P-P-G-P-A-G-G- 3.08 10.6 678A-P-P-P-A-A-A-P-G-G-A OH-4867-31_146C-C-G-P-P-G-A-A-G-P-G-C-P-P-G-P-A-G-G- 5.98 20.6 679A-P-P-P-A-A-A-P-G-G-A OH-4867-31_147C-C-G-P-P-T-A-A-G-P-G-C-P-P-G-P-A-G-G- 3.28 11.3 680A-P-P-P-A-A-A-P-G-G-A OH-4867-31_148C-C-G-P-P-C-A-A-G-P-G-C-P-P-A-P-A-G-G- 0.53 1.8 681A-P-P-P-A-A-A-P-G-G-A OH-4867-31_149C-C-G-P-P-C-A-A-G-P-G-C-P-P-C-P-A-G-G- 0.27 0.9 682A-P-P-P-A-A-A-P-G-G-A OH-4867-31_150C-C-G-P-P-C-A-A-G-P-G-C-P-P-T-P-A-G-G- 0.24 0.8 683A-P-P-P-A-A-A-P-G-G-A OH-4867-31_151C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-A-G- 0.16 0.6 684A-P-P-P-A-A-A-P-G-G-A OH-4867-31_152C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-C-G- 0.32 1.1 685A-P-P-P-A-A-A-P-G-G-A OH-4867-31_153C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-T-G- 0.21 0.7 686A-P-P-P-A-A-A-P-G-G-A

TABLE 13Single 2′-O-methyl Nap-U Substitutions, and multiple 2′-O-methyl substitutions in VEGF aptamer4867-31_143 Aptamer ID. ratio to % Activity Seq. ID. No. Sequence 5′→ 3′K_(d) (M) parent Remaining NO. 4867-31_143C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G-G-A-P-P-P-A-A- 1.4E−10 1.0 12%, 7%586 A-P-G-G 4867-31_154C-C-G-P¹-P-C-A-A-G-P-G-C-P-P-G-P-A-G-G-A-P-P-P-A-A- 5.9E−09 44 687A-P-G-G 4867-31_155 C-C-G-P-P¹-C-A-A-G-P-G-C-P-P-G-P-A-G-G-A-P-P-P-A-A-3.3E−09 24 688 A-P-G-G 4867-31_156C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G-G-A-P-P-P-A-A- 6.8E−10 5.0 52%, 74%689 A-P-G-G 4867-31_157C-C-G-P-P-C-A-A-G-P-G-C-P¹-P-G-P-A-G-G-A-P-P-P-A-A- 1.5E−09 11 54%, 690A-P-G-G 101% 4867-31_158C-C-G-P-P-C-A-A-G-P-G-C-P-P¹-G-P-A-G-G-A-P-P-P-A-A- 1.7E−09 12 59%, 691A-P-G-G 106% 4867-31_159C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P¹-A-G-G-A-P-P-P-A-A- 5.6E−10 4.1 7%, 64%692 A-P-G-G 4867-31_160C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G-G-A-P¹-P-P-A-A- 1.5E−09 11 52%, 95%693 A-P-G-G 4867-31_161C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G-G-A-P-P¹-P-A-A- 6.4E−11 0.47 5%, 4%,694 A-P-G-G −2% 4867-31_162C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G-G-A-P-P-P¹-A-A- 8.9E−09 65 84%, 695A-P-G-G 117% 4867-31_163C-C-G-P-P-C-A-A-G-P-G-C-P-P-G-P-A-G-G-A-P-P-P-A-A- 2.6E−10 1.9 53%, 45%696 A-P-G-G 4867-31_183C-C-G-P-P-C-A-A-G-P-G-C-P-P-V-P-A-V-G-A-P-P-P-A-A- 9.1E−11 0.67 15% 697A-P-G-G 4867-31_184 C-C-G-P-P-C-A-A-G¹-P-G-C-P-P-V-P-A¹-V-G-A-P-P-P-A-A-8.4E−11 0.62 7% 698 A-P-G-G 4867-31_185C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-V-P-A¹-V-G-A-P-P-P-A- 2.6E−10 1.9 50% 699A-A-P-G¹-G¹ 4867-31_186C¹-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-V-P-A¹-V-G-A-P-P-P-A- 1.6E−09 12 77% 700A¹-A-P-G¹-G¹ 4867-31_187C-C-G-P-P-C-A-A-G¹-P-G-C-P-P-V-P-A¹-G-G-A-P-P-P-A-A- 3.2E−11 0.24 −2%701 A-P-G-G 4867-31_188C-C-G-P-P-C¹-A-A-GI-P-G-C¹-P-P-V-P-A¹-G¹-G-A-P-P-P-A- 3.8E−11 0.28 −2%702 A-A-P-G¹-G¹ 4867-31_189C¹-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-V-P-A¹-G¹-G-A-P-P-P-A- 9.0E−10 6.6 13%703 A¹-A-P-G¹-G¹

TABLE 14 Multiple 2′-O-methyl substitutions in VEGF aptamer 4867-31_143Ratio to Aptamer 4867- Seq. ID. ID. No. Sequence 5′→3′ K_(d) (M) 31_188NO. 4867- C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-V-P-A¹-G¹-G-A-P-P-P- 4.8E−111.0 702 31_188 A-A-A-P-G¹-G¹ 4867-C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-V-P-A¹-G¹-G-A-P-P¹-P- 8.9E−11 1.9 70431_190 A-A-A-P-G¹-G¹ 4867-C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹-P-A¹-G¹-G-A-P-P-P- 1.0E−10 2.1 70531_191 A-A-A-P-G¹-G¹ 4867-C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹-P-A¹-G¹-G-A-P-P¹-P- 3.5E−11 0.7 70631_192 A-A-A-P-G¹-G¹

TABLE 15 PDGF homodimers of 4149-8_379 and 5169-4_26. Aptamer ID.Seq. ID. No. Sequence (5′→3′) K_(i) (pM) NO. 4149-8_438Z-M-A¹-C-H-G-Z¹-Z-A-C-C3-C¹-G-C-G-Z- 4.2 707Z-Z¹-A¹-Z-A¹-G-C-G¹-Z-M-A¹-C-H-G-Z¹-Z-A-C-C3-C¹-G-C-G-Z-Z-Z¹-A¹-Z-A¹-G-C- G¹ 4149-8_439Z-M-A¹-C-H-G-Z¹-Z-A-C-C3-C¹-G-C-G-Z- 3.5 708Z-Z¹-A¹-Z-A¹-G-C-G¹-H-Z-M-A¹-C-H-G-Z¹-Z-A-C-C3-C¹-G-C-G-Z-Z-Z¹-A¹-Z-A¹-G- C-G¹ 4149-8_440Z-M-A¹-C-H-G-Z¹-Z-A-C-C3-C¹-G-C-G-Z- 1.4 709Z-Z¹-A¹-Z-A¹-G-C-G¹-H-H-Z-M-A¹-C-H-G-Z¹-Z-A-C-C3-C¹-G-C-G-Z-Z-Z¹-A¹-Z-A¹-G- C-G¹ 4149-8_441Z-M-A¹-C-H-G-Z¹-Z-A-C-C3-C¹-G-C-G-Z- 0.78 710Z-Z¹-A¹-Z-A¹-G-C-G¹-H-H-H-Z-M-A¹-C-H-G-Z¹-Z-A-C-C3-C¹-G-C-G-Z-Z-Z¹-A¹-Z-A¹- G-C-G¹ 4149-8_442Z-M-A¹-C-H-G-Z¹-Z-A-C-C3-C¹-G-C-G-Z- 0.51 711Z-Z¹-A¹-Z-A¹-G-C-G¹-H-H-H-H-Z-M-A¹-C-H-G-Z¹-Z-A-C-C3-C¹-G-C-G-Z-Z-Z¹-A¹-Z- A¹-G-C-G¹ 4149-8_443Z-M-A¹-C-H-G-Z¹-Z-A-C-C3-C¹-G-C-G-Z- 0.25 712Z-Z¹-A¹-Z-A¹-G-C-G¹-H-H-H-H-H-Z-M-A¹-C-H-G-Z¹-Z-A-C-C3-C¹-G-C-G-Z-Z-Z¹-A¹- Z-A¹-G-C-G¹ 4149-8_444Z-M-A¹-C-H-G-Z¹-Z-A-C-C3-C¹-G-C-G-Z- 0.61 713Z-Z¹-A¹-Z-A¹-G-C-G¹-H-H-H-H-H-H-Z-M-A¹-C-H-G-Z¹-Z-A-C-C3-C¹-G-C-G-Z-Z-Z¹- A¹-Z-A¹-G-C-G¹ 4149-8_379DZ-M-A¹-C-H-G-Z¹-Z-A-C-C3-C¹-G-C-G-Z- 11 714Z-Z¹-A¹-Z-A¹-G-C-G¹-G¹-C-G-A¹-Z-A¹-Z¹-Z-Z-G-C-G-C-C3-C¹-A-Z-Z¹-G-H-C-A¹-M- Z 4149-8_445Z-M-A¹-C-H-G-Z¹-Z-A-C-C3-C¹-G-C-G-Z- 1.1 715Z-Z¹-A¹-Z-A¹-G-C-G¹-H-H-G¹-C-G-A¹-Z-A¹-Z¹-Z-Z-G-C-G-C¹-C3-C-A-Z-Z¹-G-H-C- A¹-M-Z 4149-8_446Z-M-A¹-C-H-G-Z¹-Z-A-C-C3-C¹-G-C-G-Z- 0.61 716Z-Z¹-A¹-Z-A¹-G-C-G¹-H-H-H-H-G¹-C-G-A¹-Z-A¹-Z¹-Z-Z-G-C-G-C¹-C3-C-A-Z-Z¹-G- H-C-A¹-M-Z 4149-8_447Z-M-A¹-C-H-G-Z¹-Z-A-C-C3-C¹-G-C-G-Z- 0.88 717Z-Z¹-A¹-Z-A¹-G-C-G¹-H-H-H-H-H-H-G¹-C-G-A¹-Z-A¹-Z¹-Z-Z-G-C-G-C¹-C3-C-A-Z-Z¹- G-H-C-A¹-M-Z 5169-4_134C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- 28 718P-C-P-C-G-A-C-A-G-C-A-P-G-P-A-P-G-C- A-C-A-P-C-P 5169-4_135C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- 9.3 719P-C-P-H-C-G-A-C-A-G-C-A-P-G-P-A-P-G- C-A-C-A-P-C-P 5169-4_136C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- 8.7 720P-C-P-H-H-C-G-A-C-A-G-C-A-P-G-P-A-P- G-C-A-C-A-P-C-P 5169-4_137C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- 5.1 721P-C-P-H-H-H-C-G-A-C-A-G-C-A-P-G-P-A- P-G-C-A-C-A-P-C-P 5169-4_138C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- 4.9 722P-C-P-H-H-H-H-C-G-A-C-A-G-C-A-P-G-P- A-P-G-C-A-C-A-P-C-P 5169-4_139C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- 3.6 723P-C-P-H-H-H-H-H-C-G-A-C-A-G-C-A-P-G- P-A-P-G-C-A-C-A-P-C-P 5169-4_140C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- 3.6 724P-C-P-H-H-H-H-H-H-C-G-A-C-A-G-C-A-P- G-P-A-P-G-C-A-C-A-P-C-P 5169-4_26DC-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- 11 725P-C-P-P-C-P-A-C-A-C-G-P-A-P-G-P-A-C- G-A-C-A-G-C 5169-4_141C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- 13 726P-C-P-H-H-P-C-P-A-C-A-C-G-P-A-P-G-P- A-C-G-A-C-A-G-C 5169-4_142C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- 5.5 727P-C-P-H-H-H-H-P-C-P-A-C-A-C-G-P-A-P- G-P-A-C-G-A-C-A-G-C 5169-4_143C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A- 2.0 728P-C-P-H-H-H-H-H-H-P-C-P-A-C-A-C-G-P- A-P-G-P-A-C-G-A-C-A-G-C

TABLE 16 VEGF homodimers of 4867-31_192. Aptamer ID. Seq. ID. No.Sequence (5′→ 3′) K_(i) (pM) NO. 4867-31_395C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹-P- 6.8 729A¹-G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹-C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹-P-A¹-G¹-G- A-P-P¹-P-A-A-A-P-G¹-G¹4867-31_396 C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹-P- 50 730A¹-G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹-H-C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹-P-A¹-G¹- G-A-P-P¹-P-A-A-A-P-G¹-G¹4867-31_397 C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹-P- 43 731A¹-G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹-H-H-C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹-P-A¹- G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹4867-31_398 C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹-P- 29 732A¹-G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹-H-H-H-C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹-P- A¹-G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹4867-31_399 C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹-P- 22 733A¹-G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹-H-H-H-H-C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹-P- A¹-G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹4867-31_400 C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹-P- 14 734A¹-G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹-H-H-H-H-H-C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹- P-A¹-G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹4867-31_401 C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹-P- 21 735A¹-G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹-H-H-H-H-H-H-C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹-P-A¹-G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹ 4867-C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹-P- 32 736 31_192DA¹-G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹-G¹-G¹-P-A-A-A-P-P¹-P-A-G-G¹-A¹-P-G¹-P-P-C¹- G-P-G¹-A-A-C¹-P-P-G-C-C4867-31_402 C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹-P- 27 737A¹-G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹-H-H-G¹-G¹-P-A-A-A-P-P¹-P-A-G-G¹-A¹-P-G¹-P-P- C¹-G-P-G¹-A-A-C¹-P-P-G-C-C4867-31_403 C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹-P- 20 738A¹-G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹-H-H-H-H-G¹-G¹-P-A-A-A-P-P¹-P-A-G-G¹-A¹-P-G¹- P-P-C¹-G-P-G¹-A-A-C¹-P-P-G-C-C4867-31_404 C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹-P- 33 739A¹-G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹-H-H-H-H-H-H-G¹-G¹-P-A-A-A-P-P¹-P-A-G-G¹-A¹-P-G¹-P-P-C¹-G-P-G¹-A-A-C¹-P-P-G-C-C

TABLE 17 PDGF-VEGF aptamer constructs Aptamer Seq. ID. ID. No.Sequence 5′→ 3′ NO. 4149-8_313C-C-G-P-P-C-A-A-G-P-G-C-P-P-C3-P-A-C3-G-A-P-P-P-A-A-A-P-G-G-Z-Z-A¹-C-H-G-Z-Z-A-740 C-A¹-C¹-G-C-G-Z-Z-Z-A¹-Z-A¹-G-C 4149-8_314C-C-G-P-P-C-A-A-G-P-G-C-P-P-C3-P-A-C3-G-A-P-P-P-A-A-A-P-G-G-H-Z-Z-A¹-C-H-G-Z-Z-741 A-C-A¹-C¹-G-C-G-Z-Z-Z-A¹-Z-A¹-G-C 4149-8_315C-C-G-P-P-C-A-A-G-P-G-C-P-P-C3-P-A-C3-G-A-P-P-P-A-A-A-P-G-G-H-H-Z-Z-A¹-C-H-G-Z-742 Z-A-C-A¹-C¹-G-C-G-Z-Z-Z-A¹-Z-A¹-G-C 4149-8_316C-C-G-P-P-C-A-A-G-P-G-C-P-P-C3-P-A-C3-G-A-P-P-P-A-A-A-P-G-G-H-H-H-Z-Z-A¹-C-H-G-743 Z-Z-A-C-A¹-C¹-G-C-G-Z-Z-Z-A¹-Z-A¹-G-C 4149 8_317Z-Z-A¹-C-H-G-Z-Z-A-C-A¹-C¹-G-C-G-Z-Z-Z-A¹-Z-A¹-G-C-G¹-C-C-G-P-P-C-A-A-G-P-G-C-P-744 P-C3-P-A-C3-G-A-P-P-P-A-A-A-P-G 4149 8_318Z-Z-A¹-C-H-G-Z-Z-A-C-A¹-C¹-G-C-G-Z-Z-Z-A¹-Z-A¹-G-C-G¹-H-C-C-G-P-P-C-A-A-G-P-G-C-745 P-P-C3-P-A-C3-G-A-P-P-P-A-A-A-P-G 4149 8_319Z-Z-A¹-C-H-G-Z-Z-A-C-A¹-C¹-G-C-G-Z-Z-Z-A¹-Z-A¹-G-C-G¹-H-H-C-C-G-P-P-C-A-A-G-P-G-746 C-P-P-C3-P-A-C3-G-A-P-P-P-A-A-A-P-G 4149-8320Z-Z-A¹-C-H-G-Z-Z-A-C-A¹-C¹-G-C-G-Z-Z-Z-A¹-Z-A¹-G-C-G¹-H-H-H-C-C-G-P-P-C-A-A-G-P-747 G-C-P-P-C3-P-A-C3-G-A-P-P-P-A-A-A-P-G

TABLE 18 Binding affinity and in vitro activity of PDGF-VEGF aptamerconstructs. Hs27 PDGFRβ PDGF-BB % Activity PDGF-AB VEGF VEGF121 TargetSeqID Kd (M) Remaining Kd (M) Kd (M) Kd (M) PDGF-BB 4149-8_39 8.75E−114.76E−11 1.00E−06 1.00E−06 PDGF-BB 4149-8_130 7.59E−11 1.41E−10 1.00E−061.00E−06 PDGF-BB 4149-8_273 6.14E−11 1.89E−10 1.00E−06 1.00E−06 VEGF1214867-31_51 1.26E−07 1.48E−08 1.96E−10 7.28E−10 VEGF121 4867-31_1831.95E−07 1.45E−08 1.38E−10 6.65E−10 Both 4149-8_313 9.33E−11  5%1.65E−10 2.58E−10 4.31E−09 Both 4149-8_314 1.36E−10  2% 1.11E−101.43E−10 1.55E−09 Both 4149-8_315 1.19E−10  2% 1.23E−10 8.60E−117.58E−10 Both 4149-8_316 8.97E−11  2% 1.37E−10 1.57E−10 5.00E−10 Both4149-8_317 6.38E−11 58% 8.09E−11 Both 4149-8_318 4.31E−11 43% 8.18E−112.70E−10 2.47E−09 Both 4149-8_319 2.42E−11 17% 3.02E−11 1.25E−101.07E−09 Both 4149-8_320 1.50E−11 13% 4.93E−11 7.68E−11 4.22E−10

TABLE 19 PDGF/VEGF aptamer construct 4149-8_401 Seq. Aptamer ID. ID. No.Sequence 5′→ 3′ NO. 4149-Z-M-A¹-C-H-G-Z¹-Z-A-C-C3-C¹-G-C-G-Z-Z-Z¹-A¹-Z-A¹-G-C-G¹-H-H-H-C- 7488_401 C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹-P-A¹-G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹

TABLE 20 Binding affinity of PDGF/VEGF aptamer construct 4149-8_401PDGF-BB PDGF-AB VEGF165 VEGF121 Target Seq ID Kd (M) Kd (M) Kd (M) Kd(M) Both 4149-8_320 1.50E−11 4.93E−11 7.68E−11 4.22E−10 Both 4149-8_4011.51E−11 1.93E−10 2.77E−10 3.74E−11

TABLE 22Binding affinities of PDGF/VEGF aptamer constructs comprising 5169-4_26and 4867-31_192. K_(d) Seq. Aptamer (nM) K_(d) (nM) ID. ID. No.Sequence (5′ → 3′) PDGF VEGF121 NO. 5169-4_93C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹- 0.00020 0.052 749P-A¹-G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹-H- H-H-H-H-H-C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A-P-C-P 5169-4_94 C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹- 0.000500.066 750 P-A¹-G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹-H-H-H-H-H-C-G-A-C-A-G-C-A-P-G-P-A- P-G-C-A-C-A-P-C-P 5169-4_95C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹- 0.0012 0.043 751P-A¹-G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹-H- H-H-H-C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A-P-C-P 5169-4_96 C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹- 0.00450.076 752 P-A¹-G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹-H-H-H-C-G-A-C-A-G-C-A-P-G-P-A-P-G- C-A-C-A-P-C-P 5169-4_97C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹- 0.0037 0.10 753P-A¹-G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹-H- H-C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A-P-C-P 5169-4_98 C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹- 0.0053 0.14 754P-A¹-G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹-H- C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A-C-A-P-C-P 5169-4_99 C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A- 0.0021 0.120 755C-A-P-C-P-H-H-H-H-H-H-C-C-G-P-P- C¹-A-A-G¹-P-G-C¹-P-P-G¹-P-A¹-G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹ 5169- C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A- 0.00150.056 756 4_100 C-A-P-C-P-H-H-H-H-H-C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹-P-A¹-G¹-G-A-P- P¹-P-A-A-A-P-G¹-G¹ 5169-C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A- 0.00050 0.13 757 4_101C-A-P-C-P-H-H-H-H-C-C-G-P-P-C¹-A- A-G¹-P-G-C¹-P-P-G¹-P-A¹-G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹ 5169- C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A- 0.011 0.14 7584_102 C-A-P-C-P-H-H-H-C-C-G-P-P-C¹-A-A-G¹-P-G-C¹-P-P-G¹-P-A¹-G¹-G-A-P-P¹-P- A-A-A-P-G¹-G¹ 5169-C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A- 0.0061 0.12 759 4_103C-A-P-C-P-H-H-C-C-G-P-P-C¹-A-A-G¹- P-G-C¹-P-P-G¹-P-A¹-G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹ 5169- C-G-A-C-A-G-C-A-P-G-P-A-P-G-C-A- 0.0046 0.28 760 4_104C-A-P-C-P-H-C-C-G-P-P-C¹-A-A-G¹-P- G-C¹-P-P-G¹-P-A¹-G¹-G-A-P-P¹-P-A-A-A-P-G¹-G¹

TABLE 23 Aptamers and aptamer constructs tested in Ocular PK Studies.Test Article Sequence ID Target(s) PEG SL1010 4867-31_192 VEGF 40 kDaSL1011 4149-8_379 PDGF 40 kDa SL1012 4149-8_401 VEGF and PDGF 20 kDaSL1013 4149-8_401 VEGF and PDGF 40 kDa

TABLE 24 Concentrations in the Vitreous Humor For Aptamer and AptamerConstructs in the Ocular Pharmacokinetic Studies. SL1010 SL1011 SL1012SL1013 Time EYE Con- Con- Con- Con- Point (OS or centration centrationcentration centration (hours) OD) (μg/mL) (μg/mL) (μg/mL) (μg/mL) 2 OS296 280 538 563 2 OD 296 298 682 634 24 OS 195 164 330 520 24 OD 198 165433 493 48 OS 188 131 359 376 48 OD 207 116 267 336 96 OS 146 96.3 227307 96 OD 139 97.8 204 277 192 OS 70.2 16.8 92.8 145 192 OD 82.4 18.267.8 132 OS-Oculus Sinister; OD-Oculus Dexter

TABLE 25 Vitreous Humor Half-Life for Aptamer and Aptamer ConstructsFollowing a Single Bilateral Intravitreous Dose to NZW Rabbits.Half-life 95% Confidence Test Article (hours) Interval SL1010 105 90-128SL1011 47 42-55 SL1012 69 58-85 SL1013 92 81-106

1. A second aptamer having binding affinity for a target proteincomprising a first modified C-5 position uracil and a second modifiedC-5 position uracil, wherein the first modified C-5 position uracil andthe second modified C-5 position uracil are different C-5 positionmodified uracils, wherein the second aptamer has improved bindingaffinity for the target protein compared to the binding affinity of afirst aptamer, wherein the first aptamer comprises the first modifiedC-5 position uracil without the second modified C-5 position uracil, andwherein the first modified C-5 position uracil and the second modifiedC-5 position uracil are substituted with a moiety independently selectedfrom the group consisting of

wherein, the * denotes the point of attachment of the moiety to the C-5position of the uracil; and wherein, R″″ is selected from the groupconsisting of a branched or linear lower alkyl (C1-C20); a hydroxyl(OH); a halogen (F, Cl, Br, I) and a nitrile (CN).
 2. The aptamer ofclaim 1, wherein the first aptamer and the second aptamer are DNAaptamers, RNA aptamers or a combination thereof.
 3. The aptamer of claim1, wherein the binding affinity of the second aptamer for the targetprotein is about 3-fold, 5-fold, 15-fold, 20-fold, 30-fold better thanthat binding affinity of the first aptamer for the target protein. 4.The aptamer of claim 1, wherein the binding affinity of the secondaptamer for the target protein is up to 100-fold better than thatbinding affinity of the first aptamer for the target protein.
 5. Theaptamer of claim 1, wherein the modification to the first aptamer thatresults in the second aptamer is selected from the group consisting of5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-benzylcarboxyamide)-2′-O-methyluridine,5-(N-benzylcarboxyamide)-2′-fluorouridine,5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU),5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine (ThdU),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-tyrosylcarboxyamide)-2′-deoxyuridine (TyrdU),5-(N-3,4-methylenedioxybenzylcarboxyamide)-2′-deoxyuridine (MBndU),5-(N-4-fluorobenzylcarboxyamide)-2′-deoxyuridine (FBndU),5-(N-3-phenylpropylcarboxyamide)-2′-deoxyuridine (PPdU),5-(N-imidizolylethylcarboxyamide)-2′-deoxyuridine (ImdU),5-(N-isobutylcarboxyamide)-2′-O-methyluridine,5-(N-isobutylcarboxyamide)-2′-fluorouridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-(N—R-threoninylcarboxyamide)-2′-deoxyuridine (ThrdU),5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,5-(N-tryptaminocarboxyamide)-2′-fluorouridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU),5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine,5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine),5-(N-2-naphthylmethylcarboxyamide)-2′-deoxyuridine (2NapdU),5-(N-2-naphthylmethylcarboxyamide)-2′-O-methyluridine,5-(N-2-naphthylmethylcarboxyamide)-2′-fluorouridine,5-(N-1-naphthylethylcarboxyamide)-2′-deoxyuridine (NEdU),5-(N-1-naphthylethylcarboxyamide)-2′-O-methyluridine,5-(N-1-naphthylethylcarboxyamide)-2′-fluorouridine,5-(N-2-naphthylethylcarboxyamide)-2′-deoxyuridine (2NEdU),5-(N-2-naphthylethylcarboxyamide)-2′-O-methyluridine,5-(N-2-naphthylethylcarboxyamide)-2′-fluorouridine,5-(N-3-benzofuranylethylcarboxyamide)-2′-deoxyuridine (BFdU),5-(N-3-benzofuranylethylcarboxyamide)-2′-O-methyluridine,5-(N-3-benzofuranylethylcarboxyamide)-2′-fluorouridine,5-(N-3-benzothiophenylethylcarboxyamide)-2′-deoxyuridine (BTdU),5-(N-3-benzothiophenylethylcarboxyamide)-2′-O-methyluridine, and5-(N-3-benzothiophenylethylcarboxyamide)-2′-fluorouridine.
 6. Theaptamer of claim 1, wherein the modification to the first aptamer thatresults in the second aptamer is selected from the group consisting of5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU),5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine (ThdU),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-tyrosylcarboxyamide)-2′-deoxyuridine (TyrdU),5-(N-3,4-methylenedioxybenzylcarboxyamide)-2′-deoxyuridine (MBndU),5-(N-4-fluorobenzylcarboxyamide)-2′-deoxyuridine (FBndU),5-(N-3-phenylpropylcarboxyamide)-2′-deoxyuridine (PPdU),5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-(N—R-threoninylcarboxyamide)-2′-deoxyuridine (ThrdU),5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU),5-(N-2-naphthylmethylcarboxyamide)-2′-deoxyuridine (2NapdU),5-(N-1-naphthylethylcarboxyamide)-2′-deoxyuridine (NEdU),5-(N-2-naphthylethylcarboxyamide)-2′-deoxyuridine (2NEdU) and5-(N-3-benzothiophenylethylcarboxyamide)-2′-deoxyuridine (BTdU).